ISSN:2320-8694

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Journal of Experimental Biology

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ISSN:2320-8694 ISSN:2320 -8694

Journal of Experimental Biology

AndVolume Agricultural 7 Issue I ebruary, Sciences 2019 Volume 7 Issue I ebruary, 2019

JEBAS

ISSN No. 2320 – 8694

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Volume No – 7 Issue No – III June, 2019

Journal of Experimental Biology and Agricultural Sciences

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Dr. ANIL KUMAR (Ph.D.) Asstt. Professor (Soil Science) Farm Science Centre (KVK) Booh, Tarn Taran, Punjab (India) – 143 412 Email: [email protected]

JEBAS INDEX

PROTEIN LIGAND DOCKING: A MINI REVIEW WITH A CASE STUDY 243 — 248 DOI: 10.18006/2019.7(3).243.248

IMPACT OF FERTILIZER RECOMMENDATIONS BASED ON SOIL HEALTH CARD ON FERTILIZER 249 — 254 CONSUMPTION, PRODUCTIVITY AND PROFITABILITY OF FARMERS DOI: 10.18006/2019.7(3).249.254

SYNTHESIS, CHARACTERIZATION AND APPLICATION OF CHITOSAN- G- ACRYLAMIDE COMPOSITE 255 — 265 ON SOILS OF GANGETIC PLAIN REGION DOI: 10.18006/2019.7(3).255.265

GROWTH ANALYSIS OF AREA, PRODUCTION AND PRODUCTIVITY OF WHEAT CROP IN HARYANA 266 — 272 AND INDIA DOI: 10.18006/2019.7(3).273.272

PARAMETERS OF AMMI MODEL TO ASSESS G×E INTERACTION OF MALT BARLEY GENOTYPES 273 — 280 DOI: 10.18006/2019.7(3).273.280

GROWING CONDITION INFLUENCES VARIATION IN INITIATION TO WILTING OF FLOWERS OF 281 — 288 WINTER ANNUALS DOI: 10.18006/2019.7(3).281.288

INFLUENCE OF ENVIRONMENTAL FACTORS ON THE POPULATION DYNAMICS OF CHILLI THRIPS, 289 — 294 Scirtothrips dorsalis (HOOD) AND APHID, Aphis gossypii (GLOVER) DOI: 10.18006/2019.7(3).289.294

EFFECTIVE MANAGEMENT STRATEGY OF LITTLE LEAF DISEASE IN BRINJAL (Solanum melongena 295 — 300 L.) DOI: 10.18006/2019.7(3).295.300

EFFICACY OF SILVER NANOPARTICLES SYNTHESIZED ON Commiphora gileadensis (BALSAM) 301 — 307 EXTRACT AGAINST INFECTIOUS BACTERIA DOI: 10.18006/2019.7(3).301.307

BIODEGRADATION OF THE AZO DYE (REMAZOL BLACK B) BY Pseudomonas putida ISOLATED 308 — 315 FROM TEXTILE WASTEWATER SAMPLE DOI: 10.18006/2019.7(3).309.315

DOCUMENTATION OF PLANT DIVERSITY IN MARKETS OF GOALPARA DISTRICT OF ASSAM USED 316 — 328 FOR FOOD AND GENERAL HEALTHCARE DOI: 10.18006/2019.7(3).316.328

HORMONAL AND IMMUNOLOGICAL CHANGES DURING PREGNANCY PERIOD OF COWS 329 — 334 DOI: 10.18006/2019.7(3).329.334

In vitro CYTOTOXIC EFFECTS OF HYBRID GRAPHENE OXIDE NANOCOMPOSITES (hGONCs) ON 335 — 342 CRYOPRESERVED CAPRINE WHARTON’S JELLY DERIVED MESENCHYMAL STEM CELLS (WJ-MSCs) DOI: 10.18006/2019.7(3).335.342

Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 243 – 248

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

PROTEIN LIGAND DOCKING: A MINI REVIEW WITH A CASE STUDY

Hani S.H Mohammad Ali

Department of Biological sciences, Faculty of science, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia

Received – February 18, 2019; Revision – April 24, 2019; Accepted – May 26, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).243.248

KEYWORDS ABSTRACT

Protein Ligand protein docking is a molecular technique used to predict the structure to design a particular drug. It predicts the orientation and position of small molecule (ligand) when it is bound to a protein . It can Ligands be done by using different software's available. Molecular docking generates different structure ranked Docking on scoring function. In this review, we discussed some general information regarding molecular docking with an example of NS1 protein from yellow fever virus. NS1

* Corresponding author All the articles published by Journal of Experimental Biology and Agricultural Sciences are licensed under a E-mail: [email protected]; [email protected] (Hani S.H Mohammad Ali) Creative Commons Attribution-NonCommercial 4.0

International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All rights reserved.

Protein ligand docking 244

1 Introduction to a target having a known sampling of probable conformation of

ligand in a specific channel of target in a way to establish the The technique of molecular docking may be applied for modeling adjust conformation of the complex. This may be possible by the small molecule and protein interaction (atomic level), which use of scoring function of software (Agarwal et al., 2015). There permit the characterization and performance of these small are some approaches which include the infrared spectroscopy, X- molecules and binding location to proteins. This also helps in ray crystallography and Nuclear Magnetic Resonance (NMR) interpreting important biochemical processes. Docking process spectroscopy, for the determination and creation of three- includes two major steps; first ligand conformational prediction dimensional structure of every biomolecules or organic molecules. along with location and pose while second step is assessment of Therefore, it becomes possible due to homology modeling to binding strength. These steps mainly depend on sampling investigate the possible proteins structure of unspecified structure techniques and schemes scoring respectively (McConkey et al., with high order homology to defined structure. 2002). The information of active sites can be obtained through comparative study of target protein with family of proteins having Different databases can be used for offering information on minor same function. If the information of binding sites is not available, ligand molecules. Many databases including CSD (Cambridge few programs like cavity detection or online servers can be Structural Database), ACD (Available Chemical Directory), utilized for this purpose MDDR (MDL Drug Data Report) and NCI (National Cancer Institute Database) can assist in this regard. Various interacted Understanding of drug bimolecular interactions, molecular editing conformers are produced and checked with each other, while is a striking framework, for designing normal drugs and break during performing docking. In the process of rejection, new through along with interpretation of molecule manipulation conformers are tested and still the search methodology continues (Ligand) into targeted preferred location of DNA/protein till the acceptance of one conformation. According to the (receptor) primarily to produce stable complex of significant experimental binding tendencies of docked conformers and free characteristics and with more specificity by non-covalent order energies binding appear to be more problematic than their binding orientation. Various scoring functions are engaged like consensus (Rohs et al., 2005). The information based on docking approach scoring to avoid this problem. Consensus scoring is appliance of can be integrated for free energy, binding energy and stability of number of score functions to the similar docked position to avoid complexes. Presently, the utilization of molecular docking false positive (Seeliger & Groot, 2010). For designing an effective approach to envisage the uncertain binding factors of ligand- docking protocol, lot of attempts were carried out. Certainly, receptor complex in advanced success has been achieved in the computational prediction of docking methods. The current small review article is devoted to The major aim of molecular docking is to optimize conformation latest computational techniques, emphasis has been given to types and with objective of processing less free energy binding to get and application of molecular docking to a specific case. ligand-receptor complex. The total free energy binding (ΔGbind) is exposed with the help of different factors, including hydrogen 2 Approaches of molecular docking bond (ΔGh bond), electrostatic (ΔGelec) torsional free energy (ΔGtor), dispersion and repulsion (ΔGvdw), desolvation Modeling the interaction of two molecules is an unsolved (ΔGdesolv), total internal energy (ΔGtotal) and unbound system’s problem. Various major factors of forces are existed in the inter- energy (ΔGunb). Hence, more evidences regarding the nature of molecular association, such as hydrophobic, van der walls or different sorts of connections which trigger molecular docking is stacking interactions between the aromatic/ volatile amino acids, provided by good insight of the common principle that administer hydrogen bonding and electrostatic forces. Hence with the projected free energy binding (ΔGbind) (Guedes et al., 2014). minimum information, ligand docking mimics the natural interaction of ligand with that of its receptor (Sousa et al., 2006). Data sources for the exploration of targeting with appropriate Easy methods are available for docking inflexible ligands with PDB format and a procedure to produce ligand as a PDB file are inflexible receptors and flexible ligands with inflexible receptors, required by real usage of molecular docking. In order to perform but generally, methodologies of docking taking in to account these activities certain software like Discovery studiois are conformational flexible ligands and sixty-four protein available where PDB format can be made for ligand. The ability engineering-technology and there is problem in application to relate with available target proteins/DNA these tools deliver the receptors. A search algorithm combination and functions of the organization of ligands based docking. Minor molecules docking scoring is shown in figure 1.

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245 Hani M Ali

Figure 1 Description of the docking protocol

The creation of search algorithm should have an optimal in molecular docking are (i) determination of binding mode number of entries including binding modes determined and location of ligand on a protein (ii) prediction of absolute experimentally. Though, amid two molecules, algorithm binding affinity between ligand and protein, especially in lead searching will show all probable binding modes, yet, the optimization and (iii) most important application, is to point search of this algorithm would be unfeasible due to the amount out a specific protein target in the potential drug by searching of time taken and the size of the search space. Ultimately, a database of large ligand. In previous few years, various mere amount of total conformational space may be sampled, development has been made in discovery of different scoring so an equilibrium must be attained between the amounts of the functions which have various accuracies and efficiencies in search spaced investigated and computational expense. computing. Force field, empirical, knowledge-based and Additionally, molecular dynamics, genetic algorithms, Tabu, consensus scoring are some of the common examples which and systematic searches, Monte Carlo methods, fragment- enhance efficiencies of scoring. The procedure of protein based, point complementary and distance geometry methods, docking may be divided specifically into rigid docking and are some examples of searching algorithm. Furthermore, on flexible docking (Huang et al., 2010a). the other way a many mathematical approaches in scoring function applied to forecast nature of the non-covalent 2.1 Rigid Docking association known as affinity of binding. An important problem in all computational approaches is the creation of an Avoiding any sort of flexibility, this particular kind of docking energy scoring function responsible for sharply and precisely deals the ligand and receptor both and recognizes only six degrees explain the association between ligand and protein (Jain, of translational and rotational freedom. Mostly, docking suites 2006). Three most significant applications of scoring functions used rigid body docking procedure as a first step.

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Protein ligand docking 246

2.2 Flexible Docking 3.3. Docking

Flexible docking is used to model ligand flexibility of a rigid Docking is the final step, where receptor and the interactions of protein receptor. This model used conformational space of ligand which is docked upon are tested. The scoring function ligand to model its flexibility. However, in this regard some produces a score, which is dependent on the selection of best approaches have been reported about the flexibility of protein suitable ligand. taking into account ideologically. Additionally, ligand flexibility: systematic, random or stochastic, and simulation 3.4. Results evaluation of docking approaches are the three common classes of algorithm for treating flexibility of ligand (Souse et al., 2006). Molecular Normally, docking algorithm success in forecasting a ligand docking of proteins may be more challenging due to multiple binding pose is evaluated in terms of root mean square deviation degrees of freedom and their flexibility. Huang et al. (2010b) (RMSD) between heavy atom positions observed-experimentally have described protein flexibility groups which may include soft of the ligands and the one(s) forecasted in the logarithm. The docking, side-chain flexibility, molecular relaxation and protein elasticity of system is the main problem in searching of accurate ensemble docking. pose and the searching efficiency is determined by the number of degrees of freedom present in the conformational search which is 3 General procedure for docking the main aspect (Souse et al., 2006).

Many significant scientific reports are available regarding 3.5. Software of docking and description molecular docking approaches and many publications comparing different tools of molecular docking (Taylor et al., 2002; Souse et For investigation and justifying ligand protein or protein-protein al., 2006; Huang et al., 2010a). Further, four step methodology interaction there are many algorithms available. Obtaining will be described to execute the molecular docking. successful docking technologies speed and accuracy are one of the important features in the process. Many algorithms show common 3.1. Target selection methodologies along with new extensions concentrated on gaining quick and accurate method as higher as possible. For instance, Preferably, X-ray crystallography or nuclear magnetic resonance AutoDock (Morris et al., 2009), DOCK (Kuntz et al., 1982), could be used to investigate the target structure experimentally. FlexX (Rarey et al., 1996), GOLD (Jones et al., 1997), ICM This may be getting from PDB; nevertheless, docking is (Abagyan et al., 1994), ADAM (Mizutani et al., 1994), DARWIN preferred over homology models or threading while performing (Taylor & Burnett., 2000), DIVALI (Clark., 1995), and comparison. Molprobity (Chen et al., 2010) validation software DockVision (Hart & Read, 1992) are some of the most common frequently used to evaluate the quality of model. Water docking programs. molecules removal from the cavity, stabilization of charges, missing residues filling, and generation of side chains, in 3.6 Molecular docking application to a special case accordance to the available parameters, are carried out after selecting the model. The stability and biological activeness of Biochemical reaction as it goes on before experimental part of any receptor could be used at this point. examination molecular docking may determine the possibility of this reaction. Molecular docking has reformed the results in some 3.2. Preparation and Ligand selection area of research. Especially, regarding the interaction between protein target (may be an enzyme) and small molecules (ligands) Selection of ligands type for docking is mainly depends on the which may show the forecast the activation or binding aim of docking. Databases like ZINC or PubChem, or chemsketch characteristics of drug’s to nucleic acid. This information tool might be useful for this purpose (Dias & Azevedo., 2008). determines the correlation between molecular structure and Further, determination of various filters such as total charge, cytotoxicity of drug. Keeping in view this significance the molecular weight, polar surface area, solubility, commercial researchers especially medicinal chemists are focusing to interpret availability, thresholds similarity of pharmacophores, synthetic the basic anticancer mechanism of drugs at molecular level by accessibility, and absorption, distribution of molecules, examining the nucleic acid and drugs interaction properties metabolism, excretion, and toxicology characteristics, which (Mehrotra et al., 2013; Agarwal et al., 2014). In-silico required for the reduction of molecules are also required for the observations are being carried out by medicinal chemists to proper docking. evaluate/predict their results whether the drug/compound is

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247 Hani M Ali interacting with protein/DNA. Further, when the researchers clustering results obtained from selected ligands docking into NS1 become certain that the docking program is forecasting the so-called protein. For most favorable interaction, NS1 protein showed Full interaction, then the information is used to investigate the actual Fitness of -878.36 kcal/mol and estimated ΔG of -7.12 kcal/mol. binding mode of complex that progress into drug discovery. Result represented by figure 2 shows the visualization of the Additionally, in the detection of these structural modifications in a drug would be instrumental information that may results in A sequence/structure specific binding to their target (Holt et al., 2008).

3.7 Molecular docking of NS1 protein from yellow fever virus using SwissDock server

3.7.1 Yellow fever virus NS1 protein

With high fatality rate Yellow fever virus produces a non- structure protein (NS1) and is responsible for numerous epidemics of this fever (Beasley et al., 2015). This protein contains the virulence part that is responsible for causing yellow fever. In the beginning it exists in the form of dimer but later on it becomes hexamer after translocation into reticulum lumen. This form of

NS1 is responsible for causing disease as it invade the immune system. In mature form this protein contains 352 amino acids and B is used as potential marker for the identification of this virus (Rastogi et al., 2016). NS1 protein of flavivirus shows resembles with dengue and zika virus in its structure.

3.7.1 Methods

3.7.1.1 Ligand and Receptor

The C-terminal fragment structure of the NS1 protein from yellow fever virus was obtained from the Protein Data Bank [PDB ID: 5YXA] at 2.1 Å resolution. The crystal structure of protein HET atoms of water molecules was removed to clean and only chain A was selected for docking studies. The ligand molecules JJ3297, [ZINC: 02989649] were retrieved from Figure 2 Visualization of NS1 protein interaction using Swiss Dock ZINC databases (A); Visualization of NS1 interaction using UCSF Chimera (B).

3.7.1.2 Molecular docking favorable binding of the ligands into the protein NS1.

SwissDock web server was used to perform molecular docking Conclusion available at www.swissdock.ch/docking based on the docking software. This docking web server has interface to input The protein-ligand interaction studies are important in the structure- selected structure of protein and ligand from databases, based drug design. Influenza virus non-structural protein 1 (NS1) is docking parameters change, and to visualize favorable clusters demonstrated as potential therapeutic target for antiviral therapy by online. Results can also be downloaded and viewed in freely identification of specific small-molecule inhibitors Based on the available UCSF Chimera package. results of docking studies, it has been clearly expressed that ligand molecules JJ3297 showed favorable binding with NS1 protein. 3.7.2 Results Present study, has given an insight to inhibit NS1. Further In swissdock each run Gibbs free energy (ΔG) and Full Fitness of investigations are required to get more favorable interaction into NS1. the docking evaluated. On the basis of Full Fitness and cluster Conflict of Interest formation favorable binding modes were scored. The cluster ranking performed using Full Fitness value. Figure 2 shows the The authors declare that there is no conflict of interest in this manuscript.

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Protein ligand docking 248

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Agarwal S, Jangir DK, Mehrotra R, Lohani N, Rajeswari M Mehrotra R, Jangir DK, Agarwal S, Ray B, Singh P, Srivastava (2014) A Structural Insight into Major Groove Directed Binding AK (2013) Interaction studies of anticancer drug lomustine with of Nitrosourea Derivative Nimustine with DNA: A Spectroscopic calf thymus DNA using surface enhanced Raman spectroscopy. Study. PLoS ONE 9: 104-115. MAPAN 28: 273-277.

Beasley DW, McAuley AJ, Bente DA (2015) Yellow fever virus: Mizutani MY, Tomioka N, Itai A (1994) Rational automatic genetic and phenotypic diversity and implications for detection, search method for stable docking models of protein and ligand. prevention and therapy. Antiviral Research 115:48-70 Journal of Molecular Biology 243: 310-326. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC (2010). Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, MolProbity: all-atom structure valida‐tion for macromolecular Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: crystallography. ActaCrystallographica Section D 1:12-21. Automated docking with selective receptor flexibility. Journal of Computational Chemistry 16: 2785 Clark KP (1995) Flexible ligand docking without parameter adjust-ment across four li‐gand-receptor complexes. Journal of Rarey M, Kramer B, Lengauer T, Klebe GA (1996) Fast flexible Computational Chemistry 16: 1210-1226. docking method using an incremental construction algorithm. Journal of Molecular Biology 261: 470-489. Dias R, de Azevedo WF Jr. (2008) Molecular docking algorithms. Current Drug Targets 9: 1040-1047. Rastogi M, Sharma N, Singh SK (2016) Flavivirus NS1: a Guedes IA, de Magalhães CS, Dardenne LE (2014) Receptor- multifaceted enigmatic viral protein. Virology Journal 13:131 ligand molecular docking. Biophysical Reviews 6: 75-87. Rohs R, Bloch I, Sklenar H, Shakked Z (2005) Molecular Hart TN, Read RJ (1992) A multiple-start Monte Carlo docking flexibility in ab-initio drug docking to DNA: binding-site and method. Proteins 13: 206-222. binding-mode transitions in all-atom Monte Carlo simulations. Nucleic Acids Research 33: 7048-7057. Holt PA, Chaires JB, Trent JO (2008) Molecular docking of intercalators and groove-binders to nucleic acids using Autodock and Seeliger D, de Groot BL (2010) Ligand docking and binding site Surflex. Journal of Chemical Information and Modeling 48: 1602-1615. analysis with PyMOL and Autodock/Vina. Journal of Computer Aided Molecular Design 24: 417-422. Huang SY, Grinter SZ, Zou X (2010a) Scoring functions and their evaluation methods for protein-ligand docking: recent advances and Sousa SF, Fernandes PA, Ramos MJ (2006) Protein-ligand future directions. Physical Chemistry Chemical Physics 12: 12899-908. docking: current status and future challenges. Proteins 65: 15-26.

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Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 249 – 254

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

IMPACT OF FERTILIZER RECOMMENDATIONS BASED ON SOIL HEALTH CARD ON FERTILIZER CONSUMPTION, PRODUCTIVITY AND PROFITABILITY OF FARMERS

1 1 1 2 Anil Kumar *, Navjot Singh Brar , Balwinder Kumar , HK Verma

1Farm Science Centre, Guru Angad Dev Veterinary & Animal Sciences University, Tarn Taran, Punjab 2Directorate of Extension Education, Guru Angad Dev Veterinary & Animal Sciences University, Ludhiana

Received – March 27, 2019; Revision – May 14, 2019; Accepted – June 01, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).249.254

KEYWORDS ABSTRACT

B:C ratio The present study has analysed the impact of recommendations based on ‘Soil Health Card’ on fertilizer consumption, productivity and profitability of rice-wheat cropping system of the farmers of Tarn Taran Fertilizer consumption district of Majha Region of Indian Punjab. Till 2014-15, farmers used to apply chemical fertilizers on Net returns the basis of their traditional knowledge without going for soil testing. They used to apply generally 372.3 and 156.3 kg urea and di-ammonium phosphate, respectively in wheat crop, whereas the usage of Rice-wheat cropping system said fertilizers in paddy was to the extent of 371.0 and 62.5 kg per hectare, respectively. The total cost and Soil health card of cultivation of rice-wheat cropping system was ₹73,765/-. The KVK approached the farmers and suggested them to apply fertilizers based on soil testing. After following soil test based fertilizer recommendations, fertilizer usage came down to 275 kg urea and 10 kg Zn ha-1 for paddy and 275 kg urea and 125 kg DAP ha-1 in case of wheat, indicating a net saving of ₹4,414/- per hectare per annum. Use of soil health card also improved soil health of the field resulting into additional crop yields with time. Continuous application of fertilizers based on soil test resulted in increased productivity of rice and wheat to the tune of 70.0 to 76.3 and 47.5 to 51.9 q ha-1, respectively. The respective net returns and B:C ratio of rice-wheat cropping system has also increased significantly to ₹1,24,986 and 2.80 as compared to earlier net returns and B:C ratio of ₹1,04,264 and 2.41, respectively. Because of good profits, the farmers started motivating fellow farmers to adopt soil test based fertilizer application in different crops to get more crop productivity and sustaining soil health.

* Corresponding author All the articles published by Journal of Experimental Biology and Agricultural Sciences are licensed under a E-mail: [email protected] (Anil Kumar) Creative Commons Attribution-NonCommercial 4.0

International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All rights reserved.

Impact of Fertilizer Recommendations Based on Soil Health Card on Fertilizer Consumption, Productivity and Profitability of Farmers 250

1 Introduction recommendations (both organic & inorganic sources) and

corrective measures that helps farmer to obtain good yield The high use of chemical fertilizers in Indian agriculture since green potentials and better soil health. Hence, soil testing play a critical revolution has increased the crop productivity many folds. role in ensuring balanced and efficient use of fertilizers and However, their continuous, imbalanced and indiscriminate use has sustaining soil health status and crop productivity. The soil testing also produced several adverse impacts on soil environment leading facilities for the farmers in the district under study were far away to stagnated crop yields in past decades (Sebby, 2010; Kumar et al., and no guidance on said aspect was available to the farmers. The 2016; Kumar et al., 2018). Intensive cropping system following Krishi Vigyan Kendra (KVK), Tarn Taran has approached the indiscriminate use of high analysing fertilizers are further farmers and motivated them for soil testing, after that KVK has deteriorating soil health and inducing secondary and micronutrient issued soil health card to the farmers based on soil testing, where deficiencies in soils, nutrient imbalance in soil and plants, causing recommendations on nutrients application have been provided and environmental hazards and decrease in total factor productivity now an attempt has been made to analyze the impact of fertilizer (Kumar et al., 2017). Microbial populations in soil ecosystem are recommendations based on SHC on fertilizer consumption, affecting severely due to sole and indiscriminate usage of productivity and profitability of farmers of the district. chemical fertilizer nutrients. Moreover, excessive fertilizer applications are also contaminating surface and underground 2 Materials and Methods water bodies especially by nitrate leaching (Pimentel, 1997). Above facts in turn, are inducing detrimental effects on 2.1 Study site environment, causing serious health hazards to human and animals. In India, in general, blanket fertilizer recommendations Tarn Taran is one of the border districts, lies in the North West are followed for N, P and K which rarely matches soil fertility frontier of the Punjab, India. It lies between 31o 7’ and 32o 3' need. Secondary and micro nutrients are often ignored in various North latitude and 74o 29’ and 75o 23’ in the East longitude. The cropping systems. Therefore, in present context, there is dire need climate of the district classified as tropical steppe, semi-arid and to strictly follow integrated nutrient management system and hot, which is mainly characterized by general dryness except for a supply of nutrients based on recommendations given in soil health short period during southwest monsoon season. During the card (SHC). Soil testing is a scientific method to evaluate soil summer months i.e. from April to June, weather is very hot and fertility and suggesting balanced nutritional package involving dry. The weather becomes humid and cloudy during July to organic and inorganic sources of nutrients to different crops and September. The average rainfall of the district is 482.9 mm. cropping systems (Chouhan et al., 2017). However, in past, the soil testing programme in India has unable to produce sensible 2.2 Technology transfer methodologies impression on farming community due to lack of proper soil testing facilities especially micronutrient estimation, poor In order to achieve the objective of technology transfer on soil coverage, delay in dissemination of fertilizer recommendations testing or soil test based fertilizer application and its adoption by among farmers, etc. (Biswas, 2002). Keeping in view the above the farmers of the region, different technology tools such as facts, scientists are promoting integrated nutrient management training programmes, demonstrations on soil sampling technique, (INM) concept i.e. balanced and judicious use of chemical on farm experimentation, etc. at different locations were carried fertilizers along with organic sources of nutrients (locally out and used. The farmers were motivated for soil testing and available organic manures, crop residues) and biofertilizers based many farmers follow up the soil testing. The samples so collected on soil testing to sustain soil health and crop productivity on long or received from farmers of the district were air dried, ground term basis (Mahajan & Gupta, 2009; Suri & Kumar, 2011; with wooden pestle and mortar, sieved through 2 mm sieve. The Choudhary et al., 2014). soil samples were analyzed for soil reaction (pH), electrical conductivity (EC) and organic carbon (OC), available phosphorus Fertilizer recommendations based on soil heath card has been (P), potassium (K) and micronutrients viz. zinc (Zn), copper (Cu), proven as a great step towards sustainable crop production and iron (Fe) and manganese (Mn) as per the standard procedures. soil health improvement vis-a-vis reduction in fertilizer dosage by Based on soil test results, the soil health cards were issued to the farmers. Actually, soil health card provide detailed nutrients farmers and recommendations were provided for balanced information and farmers get a well monitored report about their fertilization and integrated nutrient management. Several farmers soil and they are guided by the experts to follow recommendations started following soil test based fertilizer application in rice-wheat given in soil health card and improve soil health (Chouhan et al., cropping system, which subsequently reduced the dosage of 2017). Moreover, soil health card contains crop-wise nutrient chemical fertilizers in rice-wheat cropping system.

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251 Kumar et al.

2.3 Impact *** study and sampling farmers supplements N and P nutrients through urea and DAP, respectively. However, some farmers use single super phosphate A thorough study was undertaken to assess the knowledge levels as a source of phosphorus. Potassium application is negligible in and knowledge upgradation of the farmers about soil testing. The whole district (Kumar et al., 2017). Gypsum and zinc application study has analyzed the impact of fertilizer recommendations on before transplanting of paddy is usually done by most of the fertilizer usage, crop yields, net returns, production and monetary farmers, while few farmers practiced green manuring, besides efficiencies of the farmers’ in rice -wheat cropping system. After 3 very few farmers apply organic manure (FYM) in soil, if applied years of issuing soil health cards (i.e. soil health cards were issued rates are very less than recommended ones. Moreover, none of the during 2014-15 and impact study was conducted during 2017-18), farmers applied biofertilizers. As a whole, most of the farmers data was collected randomly from 85 farmers (n = 85) of the district apply excessive doses of nitrogenous and phosphatic fertilizers to whom soil health cards were issued. The data on fertilizer usage than the recommended ones and application of most of and crop yield before and after soil testing was recorded from said nutrients/amendments such as gypsum, zinc, sulphur without any farmers (Table 1). Two major crops of the district i.e. rice and wheat deficiency and recommendations (Kumar et al., 2017). was taken into consideration for the study. Similarly, in current study, the farmers under consideration 2.4 Economic analysis usually applied 372.3, 156.3 and 20 kg urea, DAP and others fertilizers (gypsum, MOP, Zn, etc), respectively in wheat, whereas -1 -1 -1 Gross returns (₹ ha ) = Yield (q ha ) × price of produce (₹ kg ) the use of above fertilizers in paddy were to the tune of 371.0, 62.5 and 10 kg ha-1, respectively before soil testing (Table 1). -1 -1 -1 Net returns (₹ ha ) = Gross returns (₹ ha ) - cost of cultivation (₹ ha ) However, the recommended doses of urea and DAP in wheat is 275 and 137.5 kg ha-1 (Anonymous, 2017a). Similarly, only urea -1 -1 Benefit cost ratio Gross returns (₹ ha ) @ 275 kg ha has been recommended for paddy and there is no = -1 (B:C ratio) Cost of cultivation (₹ ha ) recommendation of DAP in paddy, if already applied in wheat (Anonymous, 2017b). Beside, above fertilizers zinc sulphate @ 16 -1 The system productivity of rice and wheat crops under rice-wheat (33%) or 25 (21%) kg ha is recommended in paddy, if soils are cropping system was computed by estimating wheat–equivalent–yield deficient in said nutrient. If, recommended Zn has already applied (WEY) by the formula suggested by Ahlawat & Sharma (1993) in paddy crop then there is no need to apply Zn in wheat crop (Sadana et al., 2010). In current study, it was observed that Yield of each crop (t ha–1) × Economic value of each crop (₹ t–1) WEY= farmers under study apply fertilizers much more than . Price of rice (₹ t–1) recommended ones in both the crops (Table 1).

2.5 Production and monetary efficiencies 3.2 Impact of recommendations based on soil health card on fertilizer usage Production efficiency (PE) of rice-wheat cropping system (kg ha–1 –1 day ) was computed using following expression: After providing recommendations as per soil test, a critical

–1 interpretation of the results was provided to the farmers. The farmers Total economic yield of rice-wheat cropping system as WEY (kg ha ) PE = 365 started the follow up of fertilizer recommendations given in soil health card. It was registered that the fertilizer application in wheat comes -1 -1 System profitability in terms of monetary efficiency (ME) (₹ ha–1 down to 275.0 from 372.3 kg ha with a net reduction of 97.3 kg ha day–1) was calculated using following formula: in three years of soil testing (Table 2). Similarly, in case of paddy the

–1 Total net returns of rice-wheat cropping system (₹ ha ) Table 1 Average general fertilization practices of the farmers ME= -1 365 before soil testing (kg ha )

Input Wheat Paddy 3 Results and Discussion

3.1 Average general fertilization practices of the farmers Urea 372.3 371.0

As a whole farmers of district Tarn Taran usually apply 275-400 DAP 156.3 62.5 kg Urea, 125-175 kg Di-ammonium phosphate (DAP) ha-1 in wheat (Rabi season) and 275-325 kg urea ha-1 only in paddy/rice Others * 20 10 (Kharif season) (Kumar et al., 2017). Generally, most of the *Gypsum, MOP, Zn, etc. (n=85)

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Impact of Fertilizer Recommendations Based on Soil Health Card on Fertilizer Consumption, Productivity and Profitability of Farmers 252 fertilizer consumption reduced by 96.0 kg ha-1, it comes down to improve microbial activities, increase nutrient cycling, etc. for food 275.0 kg ha-1 against earlier dose of 371.0 kg ha-1 (Table 2). After soil and fibre production (Al-Kaisi et al., 2014). The farmers having soils testing and follow up of recommended fertilizer doses by farmers, the with low organic matter content were guided for the addition of usage of other fertilizer doses such as gypsum and muriate of potash organic manures, green manuring, etc. so that organic matter level in in case of wheat become almost negligible whereas, Zn application soils could be maintained to optimum levels. The benefits of healthy reported as such in paddy i.e. 10 kg ha-1 in paddy (no difference before soil in sustaining crop production have also been witnessed by several and after soil testing) (Table 2). After regular counselling with workers (Shah et al., 2008; Kumar, 2012; Sarkar et al., 2016). farmers, some farmers adopted soil test based fertilizer application. Actually, healthy soils increase the capacity of crops to withstand Farmers were guided to reduce fertilizer doses slowly from higher weather variability and other factors. Moreover, increased uniformity application rates to recommended ones, so that sudden decrease in of nutrient availability across the field optimizes responses to other crop yields may be avoided and a sustainable crop levels may be management inputs too. All above factors lead to healthy soil and obtained. The farmers under study follow the instructions issued and improve crop productivity. Increase in the yields of paddy, soybean reduce fertilizer doses slowly and doses reaches to recommended and maize to the extent of 19.4, 13.8 and 3.0%, respectively were also ones. Chouhan et al. (2017) also reported decrease in fertilizer registered by Chouhan et al. (2017), while studying the impact of consumption among farmers in paddy, soybean and maize after fertilizer recommendations based on soil health card among the applying fertilizer based on soil test. farmers of Madhya Pradesh.

3.3 Impact of recommendations based on soil health card on 3.4 Impact of recommendations based on soil health card on crop yields economics

Keeping all other production factors constant, after applying soil test The impact of nutrient recommendations based on soil health card based fertilizer application a good positive response on crop yield of on economics of rice and wheat crops was studied. In case of rice and wheat was recorded after three years of study (Table 3). The wheat crop, the cost of cultivation reduced by ₹ 2,492 ha-1 (Table 4). yield of wheat has been increased from 47.5 to 51.9 q ha-1 indicating Similarly, production cost of rice has been decreased by ₹1,922 an increase of 9.2%. Similarly, paddy yield rose from 70.0 to 76.3 q ha-1. The gross returns have been increased by ₹ 6,710 and 9,438 ha-1 with an increase of 9.0% following recommendations given in in case of wheat and rice, respectively following soil test based soil health card (Table 3). The increases in crop yields are obvious as fertilizer application (Table 4). Net returns and benefit cost ratio soil testing provide the actual nutrient status in the soil and led to have been increased to the extent of 25.4 and 17.0%, respectively balanced fertilization. Prior soil tests helps in managing manure and in case of wheat and 16.7 and 15.3%, respectively in paddy after fertilizer application. The balanced fertilization with time improves three years of study (Table 4). the soil physical, chemical and biological properties, which in turn provide favourable conditions for plant growth and higher crop yields. After studying the individual returns from rice and wheat crops, system productivity and profitability of rice-wheat cropping The complex biological, physical and chemical interlink of healthy system as a whole was also computed before and after soil testing soil influenced plant water availability, prevent nutrient losses, (Table 5). The rice equivalent yield increased to the tune of 9.2%

Table 2 Impact of recommendations based on soil health card on fertilizer usage (kg ha-1)

Wheat Paddy Input Before After Difference Before After Difference Urea 372.3 275.0 -97.3 371.0 275.0 -96.0

DAP 156.3 125.0 -31.3 62.5 00 -62.5

Others 20 00 -20 10 10 00 (n=85)

Table 3 Impact of recommendations based on soil health card on crop yield (q ha-1)

Average yield (q ha-1) Crop Before After Difference % increase Wheat 47.5 51.9 +4.4 9.2%

Paddy 70.0 76.3 +6.3 9.0% (n=85)

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253 Kumar et al. after following *** fertilizer recommendations of soil health card. The 3.5 Impact of recommendations based on soil health card on cost of production decreased by ₹ 4,414 ha-1 (Table 5). In term of production and monetary efficiencies percentage the cost of cultivation decreased to the extent of 6.3%.

Similarly, net returns and benefit cost ratio of rice-wheat cropping The production and monetary efficiencies of rice-wheat cropping system were increased by 9.2 and 19.5% after fertilizer system as a whole increased by 19.8 and 16.1%, respectively (Table 5). Thus, indicating enhancement in productivity as well as application based on soil test (Table 6). The increases in crop profitability of farmers of the area. yields resulted in higher production efficiency due to application of optimum or desired manure and fertilizer application to the Overall, reduction in the usage of costly fertilizer inputs by crops based on prior soil test. Similarly, monetary efficiency was farmers has been registered with time that led to reduction in higher after following fertilizer recommendations because of production cost and enhancement in profitability of the farmers. reduced expenditures and more profit margins than earlier Above results are obvious as soil testing reduce the expenditure practices. Better crop yields owing to higher nutrient uptake and on costly input, which are otherwise unnecessarily added by reduced expenditure on costly fertilizer inputs may be the possible farmers in the soil. Chouhan et al. (2017) also registered 54.8 and reason for enhanced production and monetary efficiencies in 13.3% higher net returns and B:C ratio, respectively of paddy, current study. The higher production and monetary efficiencies while studying the impact of fertilizer recommendations based on following integrated nutrient package based on soil test were also soil health card on productivity and profitability of the farmers. reported by Kumar et al. (2015) in okra-pea cropping system.

-1 Table 4 Impact of recommendations based on soil health card on economics of rice and wheat (₹ ha )

Wheat Paddy Variable Before After Benefit (₹) Before After Benefit (₹)

Cost of cultivation (₹) 36270 33778 -2492 37495 35573 -1922

Gross returns (₹) 72438 79148 +6710 105700 115138 +9438

Net returns (₹) 36168 45370 +9202 68205 79565 +11360

B:C ratio 2.00 2.34 +0.34 2.81 3.24 +0.43 (n=85)

-1 Table 5 Impact of recommendations based on soil health card on economics of rice-wheat cropping system (₹ ha )

Rice-wheat cropping system Variable Before After Difference % increase/decrease

Wheat equivalent yield (q ha-1) 117.9 128.7 +10.8 9.2%

Cost of cultivation (₹) 73765 69351 -4414 6.3%

Gross returns (₹) 178029 194337 +16308 9.2%

Net returns (₹) 104264 124986 +20722 19.8%

B:C ratio 2.41 2.80 +0.39 16.1% (n=85)

Table 6 Impact of recommendations based on soil health card on production and monetary efficiencies of rice-wheat cropping system

Rice-wheat cropping system Parameter Before After Difference % increase Production efficiency 32.3 35.3 +3.0 9.2% (kg/ha/day)

Monetary efficiency 286 342 +56 19.5% (₹/ha/day) (n=85)

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Impact of Fertilizer Recommendations Based on Soil Health Card on Fertilizer Consumption, Productivity and Profitability of Farmers 254

Conclusion Kumar A (2012) Phosphorus and rain harvested water economy through Vesicular Arbuscular Mycorrhizae (VAM) in okra pea After following fertilizer recommendations given in soil health sequence. Ph.D. Thesis submitted to the CSK HPKV, Palampur (HP). card, farmers have saved ₹ 4,414 ha-1 annum-1 (in rice-wheat cropping system). Soil health also improved resulting into Kumar A, Brar NS, Pal S, Singh P (2017) Available soil macro and additional crop yields with time. The net returns and B:C ratio micro-nutrients under rice wheat cropping system in District Tarn Taran increased to the tune of 19.8 and 16.1 %, respectively in rice- of Punjab. Ecology, Environment and Conservation 23: 229-234. wheat cropping system. The magnitude of increase in production and monetary efficiencies were 9.2 and 19.5%, respectively. Thus, Kumar A, Choudhary AK, Pooniya V, Suri VK, Singh U (2016) fertilizer recommendations based on SHC has found quite Soil Factors Associated with Micronutrient Acquisition in Crops- effective and beneficial to the farmers resulting in enhanced crop Biofortification Perspective. In: Singh U, Praharaj C, Singh S, yield, income as well as improvement in soil health. These profits Singh N (Eds.) Biofortification of Food Crops. Springer, New may further increase in future after continuous following of soil Delhi. DOI 10.1007/978-81-322-2716-8_13. test based fertilizer application due to improved soil health and Kumar A, Prakash B, Brar NS, Kumar B (2018) Potential of decreased fertilizer consumption. vermicompost for sustainable crop production and soil health Acknowledgement improvement in different cropping systems. International Journal of Microbiology and Applied Sciences 7: 1042-1055. The authors are highly thankful to ICAR-Agricultural Technology Application Research Institute, Zone I and Directorate of Kumar A, Choudhary AK, Suri VK (2015) Influence of AM– Extension Education, Guru Angad Dev Veterinary & Animal fungi and applied phosphorus on growth indices, production Sciences University, Ludhiana for financial assistance and efficiency, phosphorus–use efficiency and fruit–succulence in technical guidance, respectively for carrying out this study. okra (Abelmoschus esculentus)–pea (Pisum sativum) cropping system in an acid Alfisol. Indian Journal of Agricultural Conflict of Interest Sciences 85: 1030-1037.

The authors declare that there is no conflict of interest in this manuscript. Mahajan A, Gupta RD (2009) Role of INM in Sustainable Rice–Wheat Cropping System. In: Integrated Nutrient References Management (INM) in a Sustainable Rice-Wheat Cropping Ahlawat IPS, Sharma RP (1993) Agronomic Terminology (3rd System, Springer, Dordrecht. edn). Indian Society of Agronomy, New Delhi. Pimentel D (1997) Techniques for Reducing Pesticides: Al-Kaisi M, Douelle A, Kwaw-Mensah D (2014) Soil Environmental and Economic Benefits. Chichester, UK: John Wiley. microaggregate and macroaggregate decay over time and soil Sadana US, Manchanda JS, Khurana MPS, Dhaliwal SS, Singh H carbon change as influenced by different tillage systems. Journal (2010) The Current Scenario and Efficient Management of Zinc, of Soil and Water Conservation 69: 574-580. Iron, and Manganese Deficiencies. Better Crops- South Asia: 24-26. Anonymous (2017a) Package of practices for the crops of Punjab Sarkar MIU, Rahman MM, Rahman GKMM, Nahe UA, (Rabi 2017). Directorate of Extension Education, Punjab Ahmed MN (2016) Soil Test Based Inorganic Fertilizer and Agricultural University, Ludhiana (Punjab), 34: 1-17. Integrated Plant Nutrition System for Rice (Oryza sativa L.) Anonymous (2017b) Package of practices for the crops of Punjab Cultivation in Inceptisols of Bangladesh. The Agriculturists (Kharif 2017). Directorate of Extension Education, Punjab 14: 33-42. Agricultural University, Ludhiana (Punjab), 34: 1-15. Sebby K (2010) The Green Revolution of the 1960's and Its Impact on Biswas PP (2002) Soil testing at farmers door step. Fertilizer Small Farmers in India. In: Environmental Studies Undergraduate News 47: 21-24. Student Thesis. 10. http://digitalcommons.unl.edu/envstudtheses/10.

Choudhary AK, Rahi S, Kumar A (2014) Integrated Nutrient Shah AL, Islam MR, Haque MM, Ishaque M, Miah MAM (2008) Management in Vegetable Crops. In: Advances in Field Crop Efficacy of major nutrients in rice production. Bangladesh Journal Production (Ed. Rana et al), Venus Publishers, ISBN No. 978-93- of Agricultural Research 33: 639-645. 83168-08-8, pp 29-35. Suri VK, Kumar A (2011) Potential of Integrated Nutrient Supply Chouhan RS, Sharma HO, Rathi D, Niranjan HK (2017) Impact of soil and Soil Health Improvement in Sustainable Cropping Systems of health card scheme on farmers’ income – A case study of Kharif crops in Indian Himalayas. In: Kumar A (Ed.) Sustainable Hill Madhya Pradesh. Agricultural Economics Research Review 30: 139-141. Agriculture: An overview, Agrobios (India), pp. 123-149.

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Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 255 – 265

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

SYNTHESIS, CHARACTERIZATION AND APPLICATION OF CHITOSAN- G- ACRYLAMIDE COMPOSITE ON SOILS OF GANGETIC PLAIN REGION

Priyal Pandey1*, Mahendra Kumar Verma1, Nirmal De2

Department of Soil Science and Agricultural Chemistry, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh- 221005

Received – February 15, 2019; Revision – May 14, 2019; Accepted – June 04, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).255.265

KEYWORDS ABSTRACT

Chitosan The present study was formalized to address the effect of chitosan-grafted polyacrylamide composite on soil and therefore an innovative eco-friendly chitosan-based composite was synthesized and its probable Acrylamide effect on soil chemical and biological properties were analyzed. Chitosan grafted polyacrylamide Composite composite has been synthesized in the Department of Soil Science and Agricultural Chemistry, Institute of Agricultural Sciences, Banaras Hindu University. Synthesized Chitosan grafted polyacrylamide was Characterization characterized by Fourier transformation infrared (FTIR) spectroscopy, X-ray diffraction (XRD), Scanning electron microscopy (SEM) and Atomic force microscopy (AFM). Composite showed rough morphology Soil with an average roughness 26.17 nm, found to be partially amorphous with the presence of functional group from both acrylamide and chitosan. A lab incubation study for six months revealed that synthesized Incubation composites addition in soils (Alfisol and Inceptisol) of Gangetic plain region significantly increased soil electrical conductivity (EC), cation exchange capacity (CEC), catalase activity, dehydrogenase activity, and soil microbial biomass carbon content. Composite addition in soil did not altered soil pH, acid phosphatase activity, alkaline phosphatase activity and only urease activity in soil was found to be decreased during the incubation period. The implication of information about the effect of composite addition on soil properties is crucial for its usage and application in the soil system. This valuable information might be helpful in promoting more effective use of biopolymer-based composite rather than acrylate based composite in agriculture as a slow release carrier of fertilizers and water in the soil.

* Corresponding author All the articles published by Journal of Experimental Biology and Agricultural Sciences are licensed under a E-mail: [email protected] (Priyal Pandey) Creative Commons Attribution-NonCommercial 4.0

International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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Synthesis, characterization,and application of chitosan- g- acrylamide composite on soils of Gangetic plain region 256

1 Introduction analytical grade, and all solutions were prepared with quartz

distilled water. Superabsorbent are hydrophilic, acrylate based, crosslinked structure capable of absorbing large amount of water and have 2.1 Synthesis of chitosan-based composite distinct properties to improve soil water holding capacity, reduce drought stress, increase seed sprouting, reduce For the preparation of chitosan-based composite, method of irrigation frequency and act as best carrier of water, fertilizer Harrar et al. (2014) was followed. For this, one g (20 wt %) and pesticides to plants. Most of the superabsorbent used of chitosan (CTS) was dissolved in 30 ml (v/v) acetic acid solution in a 250 ml three-necked flask, equipped with a reflux today are generally synthetic, application of which can have condenser, a funnel, a nitrogen line, and a magnetic stirrer. adverse effect on soil. Blooming with modern technology Then 0.1 g (2 wt %) of APS was added to the CTS solution which can boost up the food production without hampering the and the resulting mixture was gently stirred for 15 minutes at harmony in ecosystem is the need of present time. In this 60oC to generate free radicals. The aqueous mixed solution of context with increasing awareness on environmental issues, acrylamide and MBA added to the flask containing chitosan biodegradable superabsorbent catches interest for potential solution. The solution was stirred vigorously for another 3 commercial application in agriculture (Cannazza et al., 2014). hours and a nitrogen atmosphere was maintained all through Superabsorbent prepared with natural materials, such as the polymerization reaction. After the polymerization reaction cellulose (Bao et al., 2011), starch (Bai et al., 2015), guar gum hydrogel composite was formed. The synthesized products (Wang et al., 2009, Thombare et al., 2018), rice husk were immersed in an excess of double distilled water, washed (Francisco et al., 2012) and chitosan(Said et al., 2018), have repeatedly and then dewatered with 500 ml of methanol for 24 attracted great attention because of their abundant resources, h. Composite was then dried in an oven at 65 oC to a constant low production-cost and biodegradability. Chitosan is a weight. Finally, the product was ground (particles size in the hydrophilic polysaccharide produced by partial deacetylation range of 40–60 mesh) and stored away from moisture, heat of chitin, it is second most abundant polysaccharide next to and light. The mechanism of composite preparation was cellulose. Chitosan posses valuable properties as shown in figure 1 and setup for the preparation of biocompatibility, biodegradability, non toxicity, amino and superabsorbent composite was shown in figure 2 (Table 1).. hydroxy group that are convenient for graft polymerization which popularize its use in agriculture sector (Harrar & Dairi, 2.2 Characterization of chitosan grafted superabsorbent 2013; Cheng et al., 2017). It has been used as a carrier for 2.2.1 FTIR Analysis fertilizer, pesticides, medicines delivery. While research interest in superabsorbent is increasing, the current level of Infrared spectroscopy (IR) of the powdered samples of knowledge does not allow a fair assessment of the pros and chitosan and synthesized polymer composite was carried out cons that will arise from the superabsorbent use in agriculture. by Perkin spectra 2 spectrophotometers. Superabsorbent As a prerequisite for such an assessment, a better samples were scanned in the region of 4000-400 cm-1 using understanding of the fate and effect of superabsorbent KBr pellets. All the spectra were recorded and analyzed to products after their application is required. The present study know the type and nature of the functional group attachment in was formulized to address the effect of superabsorbent chitosan and synthesized composite composite on soil and therefore an innovative eco-friendly chitosan-based superabsorbent was synthesized and its Table 1 Properties of Chitosan based superabsorbent probable effect on soil chemical and biological properties Chitosan based Properties were analyzed in this paper. superabsorbent pH 6.8 2 Materials and Methods Size (mm) 0.5

Chitosan (CTS, 75% degree of deacetylation and medium average Water uptake capacity molecular weight, viscosity, 200-800cps), acrylamide (AAm), Distilled water (g/g) 156.7 ammonium persulfate (APS), and N-N’- methylene bisacrylamide Tap water (g/g) 96.55 (MBA), purchased from Hi-Media company (India) were used for superabsorbent composite preparation. All reagents used were of

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257 Pandey et al.

Figure 1 Mechanism of preparation of superabsorbent composite

Cu-anode (Philips) running at 40 kV and 20 mA, scanning from 3⁰ to 50⁰ using APD (automated powder diffraction) software with the following setting of the instrument: Radiation type-Cu-Kα. Sample holder made up of aluminum. The samples were cleaned with acetone solution and filled in the space defined for the specimen. The specimen holder was inserted in the X-ray diffractometer for the analysis of the randomly oriented sample.

2.2.3 SEM

Scanning electron micrographs (SEM) produced the morphological image of superabsorbent by an electron beam and was recorded on the Zeiss EVO 18 (SEM) instrument. The accelerating voltage used was 15 kV and the applied magnification of the image was 5000 X.

It provides information about the morphology of synthesized Figure 2 Reaction setup for preparation of chitosan-g-acrylamide composite chitosan grafted composite.

2.2.4 Atomic Force Microscopy

2.2.2 XRD The surface morphology, roughness were further examined by atomic force microscopy (NT-MDT SOLVER AFM/STM). AFM XRD patterns of the chitosan and synthesized composite were images were analyzed using Nova Image Analysis 2.0software. recorded by X-ray diffractometer (XRD, Bruker D8 Advance Eco), For the AFM studies, chitosan-grafted composite was USA using Cu-Kα radiation (k = 1.54 A˚) within 3 to 80, 2 theta immobilized on glass slide by cutting thin section of hydrated range. XRD is a non-destructive method for characterization of superabsorbent, put it on glass slide cover it with cover slip, and crystalline compounds. XRD was obtained with X-ray tube having subjected to image analysis.

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Synthesis, characterization,and application of chitosan- g- acrylamide composite on soils of Gangetic plain region 258

2.3 Lab incubation experiment through Whatman no. 42 filter paper. One or two mL of the aliquot was taken into 50 mL volumetric flask. Volume was made up to 10 Alluvial soil from agricultural research farm of Banaras Hindu mL with 2 M KCl-PMA solution, and 30 mL of coloring reagent was University and Red soil from Rajiv Gandhi South Campus Barkachha, added. The flask was swirled for a few seconds and placed in a bath of Mirzapur was taken for lab incubation study. A 180 days lab boiling water. After keeping it for 30 minutes on the water bath, the incubation experiment was conducted to analyze the effect of various flasks were removed and cooled immediately in cold water for 15 contact times between superabsorbent and soil on the chemical min. Volume was made to 50 mL with water and mixed thoroughly. properties and enzymatic activity of red and alluvial soil. For this The absorbance of red color developed was measured at 527 nm using O study soil samples were pre-incubated in the dark at 25 ± 1 C for spectrophotometer (McGarity & Myers, 1967). seven days to stabilize microbial activity later 0.1 % weight of superabsorbent was added to pre-incubated soil and observations were 2.5.3 Acid and alkaline phosphatase in soil taken after 1 day, 30 days,90 days and 180 days. The results were compared with control samples in which superabsorbent was not Two grams of soil was taken in two 50 mL conical flask. Out of added. Each treatment had three replicates. The soil water-holding these two sets, one was used as a control. 0.2 mL toluene and 4 capacity (WHC) was adjusted to 50% before incubation. Throughout mL of MUB (pH 6.5 for acid phosphatase and pH 11 for alkaline the experiments, soil moisture was maintained at a constant level of phosphatase) was added to all flasks and add 1 mL p-nitrophenyl 50% WHC using WET sensor (soil moisture sensor, Vernier). phosphate solution to only 1 set of samples. The flasks of both the sets were swirled for few seconds to mix the contents. They were O 2.4 Soil chemical properties closed with the help of stopper and placed in an incubator at 37 C for 1 hour. After incubation, the stoppers were removed and 1 mL

The pH of the soil was determined by using a pH meter (Mettler of 0.5 M CaCl2 and 4 mL of NaOH was added. The flasks were Toledo, Systronics, Gujarat, India), organic carbon by titration method swirled for a few seconds. 1 mL of p- nitrophenyl phosphate was (Walkley & Black, 1934) and cation-exchange capacity (CEC) was added to the remaining (control) set of samples. Suspensions were determined using sodium acetate solution (pH 8.2) with the help of filtered quickly through Whatman No. 2 filter paper. The yellow centrifuge as described by Jackson (1973). color intensity of the filtrate was measured with a blue filter at 440 nm (Chhonkar et al., 2007). 2.5 Enzyme activity analysis 2.5.4 Catalase activity in the soil In the present experiment, the activity of five enzymes

(dehydrogenase, urease, acid,and alkaline phosphatases and catalase) Catalase activity was measured by back-titrating residual H2O2 with was estimated as mentioned below. KMnO4. Two grams of soil samples were added to 40 mL distilled water with 5 mL of 0.3% hydrogen peroxide solution. The mixture

2.5.1 Dehydrogenase activity was shaken for 20 min and then 5 mL of 1.5 mol/LH2SO4 was added. Afterward the solution was filtered and titrated by using 0.02 mol/L For estimating dehydrogenase activity 1g of air-dried soil was KMnO4 till pink endpoint appears. The reacted amount of 0.02 mol/L kept in an airtight screw-capped test tube (15 mL capacity). 0.5 KMnO4, calculated per gram of dry soil (Zhang et al., 2012), was used mL of 1% glucose solution was added in each tube. The bottom of to express the activity of catalase. the tube was gently tapped to drive out all trapped oxygen, and thus a water seal was formed above the soil. 0.2 mL of 1% 2.6 Soil microbial biomass Carbon glucose solution in each of the tubes was added to saturate the soil. The tube was incubated at 28oC for 24 hours. After Incubated soil weighed 20 g (dry weight equivalent) was taken incubation 10 mL of methanol was added and shook vigorously into a glass beaker (50 ml) and labeled (using masking tape and a and allowed to stand for 6 h. Clear pink colored supernatant was pencil). In the desiccator, some wet paper were kept at the base to withdrawn and reading was taken with a spectrophotometer at a keep humidity up and to stop soil from drying out, then glass wavelength of 485 nm (blue filter) (Casida et al.,1964). beakers with soil were kept in it and chloroform was kept in another beaker. Desiccator was sealed and a vacuum was applied 2.5.2 Urease activity so that the chloroform boils for about 2 minutes. Sealed desiccator was left for 5–10 minutes; afterward the vacuum was reapplied for Five grams of soil sample was treated with 5 mL of urea solution in a a couple of minutes boiling (just to ensure good fumigation). Then glass vial/conical flask. The bottle was capped and incubated at 37OC. desiccator was sealed and kept at 25 °C for 24 h. The soil was

After 5 hours the cap was removed and 50 mL of 2M KCl-PMA extracted with 80 ml of 0.5 M K2SO4 for 20 g fumigated soil. Soil solution was added. After addition of KCl-PMA solution bottles were microbial biomass carbon was analyzed by process of Jenkinson again capped and shaken. After one hour the solution was filtered & Ladd, (1981).

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259 Pandey et al.

2.7 Statistical analysis and characteristic peak of the amino group was around 3416 cm -1, peak at 1385 cm -1and 1089 cm -1 was due to N-H and C-O stretching Multivariate analysis was applied to compare the effect of incubation which is a feature of polysaccharide. In IR spectra of chg-g- time and soil types on soil properties. The analysis was done by SPSS acrylamide a peak at 1412cm-1 was due to the C–N stretching, which software (version 20 IBM, Chicago Inc, USA) using DMRT post hoc supported the occurrence of the grafting reaction. Furthermore, the test. appearance and intensification of the peaks at 1640 cm-1 and −1 1568cm for the primary NH2 groups confirmed that polyacrylamide 3 Results and Discussion chains grew on the chitosan backbone. The stretching peak at 3414 −1 −1 3.1 Characterization cm shifted to a longer wave number (approximately 3428 cm ) which was due to overlapping of O–H stretching of chitosan and N–H 3.1.1 FTIR analysis stretching of amide groups at poly acrylamide grafts (Kumar et al., 2012; Zhang et al., 2012; Harrar et al., 2014). FTIR spectral analysis was carried out to confirm the formation of superabsorbent composite and in order to understand the chemical 3.1.2 XRD analysis structure of chitosan-grafted acrylamide composite. The infrared spectra of chitosan and chitosan grafted acrylamide were shown in The X-ray diffraction spectra for chitosan and chg-g-Paam figure 3. It was observed that the most typical absorption band of superabsorbent were shown in figure 4. The X-ray diffractions of C=O chitosan was located at 1643 cm−1 due to partial deacetylation, chitosan showed two crystalline peak at 11.86 and 19.94. However,

Figure 3 FTIR spectra of chitosan grafted acriamide composite (a) and chitosan (b)

Figure 4 XRD of chitosan (a) and chitosan grafted acrlamide composite (b)

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Synthesis, characterization,and application of chitosan- g- acrylamide composite on soils of Gangetic plain region 260 the first peak began to disappear and the second peak appeared less 3.1.3 Morphological study by SEM and AFM intense in superabsorbent diffractogram. The reduction in crystallinity can be attributed to deformation of a hydrogen bond between OH and Surface porosity of composites has great importance on swelling

NH 2 bond of chitosan. Grafting reaction proceeds initially in the behavior of hydrogels. SEM micrographs of chitosan, the grafted amorphous region but as grafting increases crystallinity decreases network was shown in figure 5 and figure 6. Chg-g-paam network leads to an increase in the amorphous region (Kumar et al., 2012; exhibits porous structure though no porosity is observed in Samandari & Gazi,2013). This indicated that the polyacrylamide chitosan structure. It was supposed that these pores were chain has been grafted on chitosan. convenient for the penetration of water and were the sites for the

Figure 5 SEM of chitosan grafted acrylamide composite (a and b)

Figure 6 Three dimensional view of chitosan grafted acrylamide composite

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261 Pandey et al.

Table 2 Properties of soil used in study

alluvial and red soil began to decrease up to 90 days of incubation Soil Properties Alluvial Soil Red Soil with 7.23 for alluvial soil and 4.8 for red soil. pH of alluvial and red soil differed insignificantly after 180 days of incubation. The decrease pH 7.67±0.27 5.87±0.35 in pH of the chitosan composite amended soil was due to the presence -1 EC (dSm ) 0.34±0.74 0.19±0.41 of carboxylate ions in the superabsorbent which decreased the pH of Organic Carbon (%) 0.57±0.11 0.37±0.24 soil slightly (Table 2). These results were in agreement with Hady et al., 2009; Bai et al., 2010 and Shahid et al., 2012. CEC (cmol (P+) kg-1 13.8±0.62 8.30±0.51

Water holding capacity (%) 45.9±0.33 37.7±0.84 3.2.1.2 Organic Carbon content and Cation exchange capacity

Addition of chitosan-grafted composite did not alter the organic interaction of external stimuli with the hydrophilic groups of the carbon content of both red and alluvial soil. Organic carbon network (Harrar et al., 2015). The porosity of the hydrogels plays content was slightly decreased with soil incubation time although multiple rolesin enhancing the total water sorption capability and this reduction was insignificant. The organic carbon content of red the rate of response by reducing the transport resistance. Hence, it soil was found to be less than that of alluvial soil (Table 3). is one of the most important properties that must be considered in Superabsorbent amended soil showed increase cation exchange the hydrogel structure. capacity with an increase in the period of incubation. Initially, CEC of alluvial soil was 13.8 CmolP+ Kg -1 soil and red soil AFM image of chitosan-grafted superabsorbent was presented in was8.5CmolP+ Kg -1 soil. After treating the soil with chitosan figure 7. AFM results were in agreement with the SEM result that grafted composite the CEC of soil was found to be increased. chitosan grafted composite was porous and rough which helped in Highest CEC was found in composite treated soil after 180 days penetration of water and nutrients. Root mean square roughness of of incubation. After addition of chitosan-grafted composite has composite was 33.91 nm, average roughness was 26.17 nm and increased CEC of soil, it may be due to the addition of chitosan- area peak-to-valley height was 295.50 nm. The result was in grafted composite had altered the cation-anion ratio of ion, zero agreement with the result of Nagarpita et al. (2017). AFM result point charge of soil opened up the negative sites in the soil thus also depicts porous behavior of composite. More rough and cation exchange capacity of soil increased (Schneider et al., porous the surface of the composite, more water could penetrate 2013). Polyacrylamide is an anionic polymer grafted on cationic the composite resulting in higher water absorbency. chitosan. As negative charge in soil increases affect the zero-point Effect of chitosan grafted composite on soil pH has been studied. charge of soil thus affected pH depended on charges in turn Red soil had lower pH as compared to alluvial soil. pH of both increased CEC of both soil.

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Synthesis, characterization,and application of chitosan- g- acrylamide composite on soils of Gangetic plain region 262

Table 3 Effect of polymer composite on chemical properties of soil

Cation exchange capacity Time pH Organic Carbon (%) + -1 Treatment (CEC) (C mole P kg ) (Days) Alluvial soil Red soil Alluvial soil Red soil Alluvial soil Red soil Control 7.73c 5.70bc 0.50a 0.37a 13.80a 8.53a 1 Composite treated soil 7.63bc 5.63bc 0.49a 0.36a 13.63a 7.96a

Control 7.63bc 5.60bc 0.47a 0.35a 13.60a 8.23a 30 Composite treated soil 7.36ab 5.36bc 0.48a 0.35a 15.76b 10.23b

Control 7.70bc 5.30b 0.45a 0.33a 13.93a 8.30a 90 Composite treated soil 7.23a 4.80a 0.48 a 0.35a 15.86b 9.30ab

Control 7.66bc 5.26b 0.46a 0.34a 14.06a 9.26ab 180 Composite treated soil 7.63bc 5.26b 0.47a 0.36a 16.76c 11.73c Means are the average of three replicates. Means followed by a common letter are not significantly different at the 5% level based on Duncan’s Multiple Range Test (DMRT).

Table 4 Effect of polymer composites on biological properties of soil

Catalase activity Dehydrogenase Acid phosphatase Alkaline Urease activity Soil microbial Time Phosphatase biomass carbon Treatment (Days) Alluvial Red soil Alluvial Red soil Alluvial Red soil Alluvial Red soil Alluvial Red soil Alluvial Red soil soil soil soil soil soil soil Control 0.6a 2.36a 43.30b 21.46b 48.30d 28.56b 184.26a 87.26a 207.16a 184.4f 186.83a 137.93b 1 Composite 0.5a 2.63a 42.76b 21.50b 47.56d 28.46b 185.86a 88.30a 208.06a 188.66f 187.73a 139.23b treated soil

Control 2.97b 4.80a 40.53b 19.82ab 53.18e 31.75b 237.16bc 141.90d 242.47b 167.09e 257.50d 170.39e 30 Composite 4c 8.26b 72.26c 21.50c 61.06f 45.13c 273.46cd 153.70e 278.42e 167.56e 252.16d 162.51d treated soil Control 6.77d 15.46d 36.06a 19.03ab 21.66b 17.63a 205.10ab 122.23c 244.44c 152.96d 232.51c 155.32c 90 Composite 8.5e 23.56e 86.70d 45.43d 35.86c 52.03c 248.73bc 167.56f 253.03d 116.33a 294.60e 190.77f treated soil Control 8.46e 12.23c 33.86a 16.60a 15.00a 15.43a 182.53a 100.93b 237.27b 141.13c 214.57b 118.90a 180 Composite 9.53f 16.80d 94.63e 70.03e 32.03c 72.96d 281.60d 176.76g 207.16a 126.00d 322.90f 221.40g treated soil Means are the average of three replicates. Means followed by a common letter are not significantly different at the 5% level based on Duncan’s Multiple Range Test(DMRT).

3.2.2 Soil biological properties Dehydrogenase was found to be increased significantly with respect to time in both soils. Dehydrogenase and catalase activity represent 3.2.2.1 Catalaseactivity and Dehydrogenase activity good health of soil (Varennes et al., 2010). As chitosan and acrylamide act as a carbon source for aerobic microbes thus Catalase activity is a representative of good aerobic microbial microbial population increases which increases enzymatic activity activity in soil. Addition of chitosan-grafted composite in soil. Dehydrogenase is an intracellular enzyme involved in the superabsorbent improved catalase activity of soil. Chitosan energy transfer in the respiration chain, and its activity typically grafted composite treated soil showed highest catalase activity occurs in all intact viable microbial cells. Thereby, its measurement after 180 days in alluvial soil. Red soil amended with chitosan is usually related to the presence of viable microorganisms and composite showed highest catalase activity after 90 days of could be considered as an index of their oxidative capability (Siafu, incubation and then decreased after 180 days of incubation in the 2017). The significant increase in dehydrogenase activity in soil composite (Table 4). samples applied with chitosan grafted composites indicated

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263 Pandey et al. microbes used chitosan grafted acrylamide as a carbon and nitrogen after decomposition it releases CO2, H2O and nitrogen, providing source for carrying out their metabolic activity and attacked cross food and energy source for the microorganism. So soil microbial linked structure converting the insoluble polymer into a soluble biomass of superabsorbent amended soil was found to be more. polymer. Thus, microbial activity increases and as a result The studied soil biological and biochemical parameters are dehydrogenase activity also increased. connected with other soil properties. Soil organic matter is one of the important factors in soil enzyme activity (Sharma et al., 2017). 3.2.2.2 Alkaline and Acid Phosphatase activity Soil enzymatic activity can be sensitive biological markers and

are influenced by many factors, e.g. soil moisture, temperature, Acid phosphatase activity was found to be highest after 30 days of aeration, pH, organic matter quality and quantity, the presence of incubation in composite amended alluvial soil (60.06 µgkg-1). inhibitors and activators. Microbial biomass C is more sensitive After 30 days acid phosphatase activity began to decrease in than total organic C to indicate soil changes because it is related to alluvial soil. Chitosan grafted amended red soil showed the soil microorganisms that are sensitive to soil variations (Sharma et highest acid phosphatase activity after 180 days of incubation i.e. al., 2017). As chitosan grafted potassium composite act as a food 72.96 µgkg-1. Alkaline phosphatase increased significantly in both substrate for microbes improved microbial biomass carbon also. soil with increasing period of incubation. Phosphatase enzyme is activated when there is low P availability in soil and phosphatase Conclusion was inhibited by inorganic phosphate, which produces feedback inhibition of this enzyme. These results are in agreement with Chitosan grafted superabsorbent altered physicochemical and Saied et al. (2016) who reported the addition of rice straw based biological property of soil.It improved the enzymatic activity of the superabsorbent increased dehydrogenase and phosphatase activity soil, increases cation exchange capacity and electrical conductivity in soil. Phosphatase activity plays an essential role in the and slightly lowers the pH of the soil. Thus present study showed no mineralization of organic P and this enzyme is activated when P adverse effect of chitosan-grafted composite addition on studied soil availability is low. In general, chitosan grafted composite treated properties. Chitosan is a biopolymer and its use in superabsorbent soils showed significantly higher phosphatase activity than control preparation can improve various qualities of the superabsorbent soil suggesting a higher amount of available substrates in composite in an eco-friendly manner. However, changes varied composite treated soils (Mahdavinia et al., 2009). according to the type of soil and dominant microbes present in soil. Effect of superabsorbent composite on microbial community is 3.2.2.3 Urease activity needed to be study more deeply for effective use of superabsorbent composite in agriculture. Urease activity was found to be decreased in both red and alluvial soil significantly. Urease activity decreased with the Acknowledgment + addition of the end product of the enzymatic reaction (NH4 ). The activity of this enzyme activity was impaired by The senior author is indebted to the Indian Council of Agricultural polyacrylate polymers containing ammonium as a counter ion Research, New Delhi, for granting funds in an extramural research (Parvathy et al., 2014). As applied chitosan grafted project on “Innovative nanoclay polymer composites for the polyacrylamide superabsorbent had amide as a main constituent promotion of rainfed agriculture” (BHU project code M- releasing ammonium ion on decomposition. Due to this reason 21/144).We are thankful to the Department of Chemistry, Institute urease activity was decreased by application of superabsorbent. of Science, BHU for FTIR and XRD analysis, CIFC,IIT, BHU for This probably resulted due to the inhibition of urease produced SEM and AFM study. + by the nitrogen that fabricated under conversation of NH4 (Sarapatka et al., 2006; Giroto et al., 2017). Abundant microbial Conflict of interests activity in the fertilized soil creates a big request for nitrogen + There is no conflict of interest. and thus, higher conversion of NH4 , which may induce a reduction in the urease activity (Faissal et al., 2017). Author contribution 3.2.3 Soil microbial biomass carbon This work was carried out in collaboration between all the Soil microbial biomass carbon increased in both alluvial and red authors. Author Nirmal De designed the study and guided in the soil in incubation experiment. The result was found to be discussion of results. Author PriyalPandey and MahendraVerma significant after the 3rd month of incubation and found to be ran the experiment, generated data, managed the literature highest after sixth months of incubation. It can be due to searches and produced an initial draft. All authors read and decomposition of chitosan-grafted superabsorbent composite, approved the final manuscript

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Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 266 – 272

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

GROWTH ANALYSIS OF AREA, PRODUCTION AND PRODUCTIVITY OF WHEAT CROP IN HARYANA AND INDIA

Nisha1*, Mohit Nain2, D.R. Aneja3

Department of Mathematics and Statistics, CCS Haryana Agricultural University, Hisar-125004 (Haryana), India

Received – February 09, 2019; Revision – April 21, 2019; Accepted – May 21, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).266.272

KEYWORDS ABSTRACT

Wheat The study intends to scrutinize the performance of wheat production in Haryana and India during the period 1966-67 to 2012-13; this time period were divided in five sub periods i.e. P-I (1966-67 to 1975- Instability 76), P-II (1976-77 to 1985-86), P-III (1986-87 to 1995-96), P-IV (1996-97 to 2005-06) and P-V (2006- Coefficient of variation 07 to 2012-13). The study is exclusively based on secondary data. The Result of study revealed positive trends in area, production and yield of wheat for both Haryana and India. Similar results were obtained Linear growth rate on the triennium basis. The contribution of area and yield in the production of wheat are comparable for both Haryana and India. All the selected three aspects (i.e. area, production and yield) have shown Compound growth rate positive growth rates in Haryana while in case of India, positive growth rates were observed for only area and production except in Period- IV (1996-97 to 2005-06). Trend of yield in India was similar to Variability that of Haryana. Similar results are observed for coefficient of variation and stability indices in production and yield of wheat in Haryana as well as India.

* Corresponding author All the articles published by Journal of Experimental Biology and Agricultural Sciences are licensed under a E-mail: [email protected] (Nisha) Creative Commons Attribution-NonCommercial 4.0

International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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Growth analysis of area, production and productivity of wheat crop 267

1 Introduction To study the contribution of area, yield and the interaction of area

and yield towards increasing the wheat production in state as well Wheat (Triticum aestivum) is one of the most important crop in as country, a decomposition analysis was performed as given the world and gives a major contribution in food grain production. below. Several workers used this decomposition analysis and It is the staple food for most of world population and an excellent studied growth performance of crops (Bastine & Palanisami, source of carbohydrates and energy without fat. Indian economy 1994; Bhatnagar & Nandal, 1994; Gupta & Saraswat, 1997; Singh in general and the economy of Haryana in particular, witnessed a & Ranjan, 1998; Siju & Kombairaju, 2001). distinctive change since mid-sixties with the development of High

Yielding Varieties (HYVs) of wheat. Abraham & Raheja (1967) ΔQ = A0 ΔY + Y0 ΔQ + ΔAΔY found that the growth in productivity of rice and wheat during 1951-52 to 1964-65 was mainly due to increase in fertilizers for The first term on the right hand side can be considered as the yield both the crops in India. Singh & Supriya (2017) studied the trend effect, the second term as the area effect and third as the in area, production and yield of wheat crop in Uttar Pradesh, India interaction effect. Thus, the total change in production can be for the period 1971-2010 and found that among the three aspects decomposed into three effects, viz.; yield effect, area effect and (area, production and productivity), productivity has shown higher the interaction effect due to the change in yield and area. growth rates due to increased trend in growth rates of area and To measure the magnitude of Variability in area, production and productivity and also for estimation of these three aspects cubic productivity, the coefficient of variation was computed using the model was found to be the best for wheat crop in India. Kumar et formula: al. (2017) analyzed growth rates in area, production and S.D Coefficient of Variation (C.V.) = × 100 productivity of major cereal crops in Sikkim and observed a X drastic reduction in the production and area of cultivation of major Where, S.D = Standard Deviation of area/production/yield cereal crops in Sikkim. Keeping this in view, the present paper is an attempt to examine the growth and instability in area, 푋 = Mean value of area/production/yield of the crop production and productivity of wheat crop in Haryana and India. under consideration Thus for examining the growth rates both linear as well as exponential functions are fitted. In order to determine the Cuddy-Della Valle Index (CDI) = C.V. 1 − 푅2 variability and Instability among the three aspects i.e. area, production and productivity coefficient of determination and Where, Cuddy-Della Valle Index are computed. C.V. = Coefficient of Variation R2 = ESS/TSS i.e. ratio of explained variation to total variation. 2 Materials and Methods ESS = Variation explained by explanatory variable. TSS = Total Variation The study was entirely based on secondary data. The time series data related to area, production and yield of wheat in Haryana 3 Results and Discussion and India for the period of 47 years i.e. 1966-67 to 2012-13 was obtained from various issues of Statistical Abstract of Haryana, Changes in area, production and yield of wheat in Haryana and India Statistical Abstract of India (http://esaharyana.gov.in/en- during study period are shown in Table 1 and Table 2 respectively. us/State-Statistical-Abstract-of-Haryana) and Agricultural For wheat crop, the area was increased from 743 to 2497 thousand Situation in India (https://www.indiaagristat.com/agriculture- hectares i.e. 1754 thousand hectares more area came under Haryana data/2/agricultural-area- land-use/152/stats.aspx). The entire study during the last 47 years ( Table 1) whereas for India the area under period (1966-67 to 2012-13) was divided into five sub-periods in wheat crop increased by 17.16 million hectares i.e. from 12.84 to order to have a period-wise examination of growth and variability 30.00 million hectares in the same period (Table 2). It depicts that rise patterns of area, production and productivity of wheat and the sub in area of wheat was 236.07 and 133.65 per cent for Haryana and periods were as follows: Period I, 1966-67to 1975-76; Period II, 1976- India respectively. So increase in area for wheat in Haryana was much 77 to 1985-86; Period III, 1986-87 to 1995-96; Period IV, 1996-97 to higher as compared to that in India. In Haryana, the production of 2005-06; Period V, 2006-07 to 2012-13. By taking wheat increased by 949.8 per cent whereas the corresponding increase area/production/yield as dependent variable (Yt) and year number (t) was 720.48 per cent at the country level during this period. So, the rise as independent variable linear as well as exponential functions were in production of wheat in Haryana was higher than that for India. The fitted for computing various growth rates. The Coefficient of average yield in Haryana shot up from 1425 kg/ha to 4452 kg/ha, determination (R2) was also computed to scrutinize whether or not the giving a net increase of 3027 kg/ha whereas for India it has increased linear and exponential functions adequately fit the available data. from 887 kg/ha to 3117 kg/ha i.e. a net increase of 2230 kg/ha (Table 1).

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268 Nisha et al.

Table 1 Changes in area, production and yield of wheat in Haryana from 1966-67 to 2012-13 on yearly and triennium basis

Yearly Basis Triennium Basis Base year Current year Net Per cent Variable Base Triennium Current Triennium Net per cent (1966-67) (2012-13) Change Change (1966-67 to (2010-11 to Change Change 1968-69) 2012-13) Area 743.00 2497.00 1754.00 236.10 827.33 2511.33 1684.00 203.55 (‘000 ha) Production 1059.00 11117.00 10058.00 949.80 1342.00 11811.00 10469.00 780.10 (‘000 tonnes) Yield 1425.00 4452.00 3027.00 212.40 1612.67 4702.00 3089.33 191.57 (kg/ha)

Table 2 Changes in area, production and yield of wheat in India from 1966-67 to 2012-13 on yearly and triennium basis

Yearly Basis Triennium Basis Current Base year Current year Variable Base year per cent Net per cent year Net Change (1966-67 to (2010-11 (1966-67) Change Change Change (2012-13) 1968-69) to 2012-13) Area 12.84 30.00 17.16 133.65 14.60 29.64 15.04 103.01 (m ha) Production 11.39 93.50 82.11 720.48 15.52 91.75 76.23 491.17 (m tonnes) Yield (kg/ha) 887.00 3117.00 2230.00 251.41 1053.00 3094.00 2041.00 193.83

On triennium basis, average area under wheat crop in Haryana Table 3 Contribution of different factors in the production of inflated from 827.33 thousand hectares (1966-67 to 1968-69) to wheat in Haryana and India 2511.33 thousand hectares (2010-11 to 2012-13) i.e. giving an Wheat increase of 203.55 per cent whereas at country level, it increased Period Effect from 14.60 million hectares to 29.64 million hectares with a net Haryana (%) India (%) change of 15.04 million hectares or 103 per cent (Table 1 and Area 50.30 38.70 Table 2). The percentage change in production of wheat was at I Yield 30.10 38.50 the tune of 780.0 per cent in Haryana and it increased by 491.17 per cent in India during this period. Increase in production of Yield × Area 19.60 22.80 wheat crop was observed more in Haryana than in India. Yield Area 28.00 16.00 shot up from 1612.67 kg/ha to 4702.0 kg/ha giving a net change of 3089.33 kg/ hectare in Haryana (191.57 per cent) whereas at II Yield 57.00 76.50 country level, it increased from 1053.0 kg/ hectare to 3094.0 Yield × Area 15.00 7.50 kg/ha showing a net change of 2041.0 kg/ hectare (193.83 per Area 24.00 20.20 cent). Contribution of different factors in the production of wheat in Haryana and India are shown in Table 3. III Yield 68.60 73.80

Yield × Area 7.40 6.00 In Haryana during Period-I, the production of wheat was increased by 30.1 and 50.3 per cent due to yield and area Area 108.00 25.00 respectively whereas their joint effect has 19.6 per cent IV Yield -7.00 26.50 contribution. At the country level, the yield, area and their Yield × Area -1.00 48.50 interaction have contributed 38.5, 38.7 and 22.8 percent respectively, towards its production (Table 3). The contribution Area 48.20 30.70 of yield was more prominent in Haryana and the contribution of V Yield 49.20 64.70 area and yield were at par in India for this period towards the Yield × Area 2.60 4.60 production of yield. Overall pattern of influence of these three components was same for both Haryana and India in the decade Area 24.90 18.50 from 1996-97 to 2005-06. During the Period-II, the production Overall Yield 22.40 34.90 was increased by 57 per cent, 28 per cent and 15 per cent due to Yield × Area 52.70 46.60 yield, area and their interaction respectively in Haryana. At the

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Growth analysis of area, production and productivity of wheat crop 269 national level, the contribution towards production by these production of wheat were 49.2 and 48.2 per cent respectively while components was 76.5 per cent, 16 per cent and 7.5 per cent only 2.6 per cent contribution was observed from their interaction. Similarly, for India, the yield and area effects were 64.7 and 30.7 per respectively. So the pattern of influence of these factors was similar both for Haryana as well as India during this period. cent respectively and 4.6 per cent due to their interaction. Hence yield was the major contributor in the production of wheat in Haryana as In Period-III, the overall production of wheat in Haryana, the yield well as in India during this period. contributed 68.6 per cent, area has 24 per cent contribution and the joint effect of area and yield was having 7.4 per cent contribution. For the overall period under study, the percentage contribution of yield, Similar results were obtained by Laitonjam et al. (2018) in the area and their interaction towards the overall production in Haryana production of pulses in India during the study period i.e. 1980-2014. were 22.4, 24.9 and 52.7 per cent respectively whereas at the country At the country level the rise in production of wheat was 73.8 per cent level the contributions of these three factors were 34.9, 18.5 and 46.6 per due to yield, 20.2 per cent due to area and 6.0 per cent from their joint cent respectively. Among the two factors of production, the contribution effect. Hence for both Haryana and India, yield played a significant of area was more in Haryana whereas the yield contributed more at the role to increase its production and the pattern of contribution was the national level for the overall period. The contributions of the joint effects similar both for Haryana and India. During Period-IV, while yield and of area and yield was at par both for increase of the production of wheat its interaction with area has small but negative effects to the extent of crop in Haryana as well in India during this period. 7 and 1 per cent respectively, only area was the factor responsible for the production of wheat crop in Haryana. At the country level, 26.5, Under wheat crop 5.33 compound growth rates was observed in 25 and 48.5 per cent contributions were observed due to yield, area Haryana which was more than India (4.18) for Period-I, while the and their interaction, respectively, towards the production of wheat. In compound growth rates for production and yield i.e. 7.87 and 3.54, Haryana during Period-V, the contribution of yield and area in the were more in case of India (Table 4). The annual increase in area,

Table 4 Growth rates of area, production and yield of wheat crop in Haryana and India

Haryana India Period Variable Linear Compound Linear Compound growth rate (b) growth rate (%) growth rate (b) growth rate (%) I Area 51.44** 5.33** 0.68** 4.18** Production 107.00** 6.64** 1.47** 7.87** Yield 20.28 1.24 40.18** 3.54** II Area 48.00** 3.15** 0.29** 1.29** Production 257.02** 7.03** 2.01** 5.48** Yield 90.85** 3.76** 68.10** 4.13** III Area 27.81** 1.49** 0.26** 1.08** Production 277.46** 4.61** 2.11 3.98 Yield 98.87** 3.07** 63.11** 2.87** IV Area 29.28** 1.36** -0.05 -0.18 Production 127.03 1.55 -0.05 -0.06 Yield 6.84 0.19 2.69 0.11 V Area 19.14** 0.78** 0.39** 1.37** Production 318.14* 2.92* 3.28** 3.94** Yield 94.22 2.11 73.50** 2.53** Overall Area 36.70** 2.25** 0.28** 1.25** Production 237.12** 4.77** 1.62** 3.68** Yield 70.44** 2.46** 45.96** 2.40**

*significant at 5% **significant at 1%

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270 Nisha et al. production and productivity for Haryana were 51.44 thousand ha, For the entire period, the trend in area under wheat crop revealed a 107.00 thousand tonnes and 20.28 kg/ha, as compared to 0.68 m ha, highly significant growth of 2.25 per cent and for its production 1.47 million tonnes and 40.18 kg/ha at the country level, respectively. and productivity it was 4.77 and 2.46 per cent respectively in Similar results were obtained by Bhatnagar & Sexena (2000) for Haryana (Table 5). Similar results were retrieved by Gautam & wheat crop in Haryana. During Period-II the values of trend Sisodia (2018) for the area, production and yield of wheat crop in coefficients were 3.15, 7.03 and 3.76 per cent for area, production and Uttar Pradesh. For India, the trend coefficient for wheat crop have yield of wheat respectively, giving an increase of 48 thousand shown positive growth rates of 1.25, 3.68 and 2.40 per cent for hectares, 257.02 thousand tonnes and 90.85 kg/ha respectively every area, production and yield respectively during this period (Table year in Haryana. Area, production and yield were increased with 5). Similar results were obtained by Ganjeer et al. (2017) for the highly significant growth rates of 1.29, 5.48 and 4.13 per cent area, production and productivity of wheat crop in different respectively and their annual increase were recorded to be 0.29 million districts of Northern Hills of Chhattisgarh for the period 1979-80 hectares, 2.01 million tonnes and 68.10 kg/ha respectively for India. to 2012-13 and Abid et al. (2014) for sugarcane in Khyber Thus the rate of increase in the yield of wheat in India (40.18 kg/ha) Pakhtunkhwa during 1980-81 to 2011-12. Hence area, production was faster than that of Haryana (86.10 kg/ha) during Period-II. and productivity of wheat have increased with highly significant growth in both Haryana and India. There was appreciable increase For Period-III, the area of wheat in Haryana escalated with highly in the productivity of wheat in India (45.96 kg/ha) as compared to significant growth rates i.e. 1.49 per cent (showing that 27.81 Haryana state (30.70 kg/ha). thousand hectares more area came under wheat crop every year). There was appreciable increase in its production with highly The coefficient of variation with respect to three components i.e. significant growth rate of 4.61 per cent giving an increase of area, production and productivity of wheat crop were higher in 277.46 thousand tonnes per annum. Comparable results were Haryana as compared to India in all the periods and the overall obtained for wheat crop in Haryana by Bhatnagar & Nandal period except for yield in period-II, area and production in period-V, (1994) and in Chattisgarh district of Madhya Pradesh by Ali & showing thereby that wide variation has occurred in Haryana as Singh (1995) for the same crop. Yield has also increased with compared to the country with respect to these parameters (Table 5). highly significant rate of 3.07 per cent (an annual increase of Similarly, instability indices were also higher in Haryana as 98.87 kg/ha). At the country level, the production of wheat in this compared to the country in all the sub-periods and overall period period has a growth rate of 3.98 per cent (i.e. 2.11 million tonnes except in production in period II and area in period-V. more production every year). Area and yield has also highly In Haryana, highest coefficient of variation in area under wheat significant growth rates of 1.08 and 2.87 per cent respectively crop was 16.76 percent in period-I followed by 10.05 percent affecting annual increase of 0.26 million hectares in area and during Period-II while least variation has occurred in its area i.e. 63.11 kg/ha yield of wheat in the country during this period. 2.00 percent during Period-V. There was decreasing trend in the coefficient of variation in area as we move from Period-I through In Haryana wheat crop have shown an appreciable increase in area Period-V both in Haryana and all India level. Similar trends were with significant rate of 1.36 percent during Period-IV while this observed for coefficient of variation and stability indices in improvement were 1.55 and 0.19 percent for its production and production and yield of wheat in Haryana as well as India. yield respectively. At the national level, area and production of wheat crop declined with growth rate of 0.18 and 0.06 per cent in Conclusions India though the yield has increased with 0.11 per cent rate of growth (an annual increase of 2.69 kg/ha). During Period-V the The study reveals positive trends in area, production and yield of area under the wheat crop has grown with significant rate of 0.78 wheat for both Haryana and India. The contributions of area and per cent, production increased significantly with 2.92 per cent yield in the production of wheat crop were comparable for both growth rate and yield increased with rate of 2.11 per cent in Haryana and India. Similar results were obtained on triennium Haryana. Similar results were obtained by Netam & Sahu (2017) bases. All the three aspects (i.e. area, production and yield) have for the area and productivity of rice crop in Chhattisgarh during shown positive growth rates in Haryana while in case of India, the period of study 2000-01 to 2011-12. positive growth rates were observed for both area and production except in Period- IV from 1996-97 to 2005-06. For the entire The growth rate for area, production and productivity during this period under study the trend of area under wheat crop revealed a period were 1.37, 3.94 and 2.53 per cent respectively giving an highly significant growth of 2.25 percent and for its production annual increase of 0.39 million hectares, 3.28 million tonnes and and productivity it was 4.77 and 2.46 percent respectively in 73.5 kg/ha respectively in India. Haryana. For India the trend coefficient for wheat crop have Shown

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Growth analysis of area, production and productivity of wheat crop 271

Table 5 Coefficient of Variation and Instability Index of Wheat Crop in Haryana and India

Haryana India Period Variable Coefficient of Instability Coefficient of Instability R2 (%) R2 (%) Variation (%) Index Variation (%) Index

Area 76.69 16.76 8.09 77.19 13.29 6.35

I Production 51.02 24.09 16.90 71.34 23.74 12.70

Yield 8.07 12.93 12.40 60.14 12.63 7.97

Area 86.54 10.05 3.69 64.07 4.86 2.91

II Production 95.19 21.20 4.65 91.55 16.69 4.85

Yield 86.96 12.53 4.52 91.98 12.82 3.63

Area 78.93 5.04 2.31 71.89 3.85 2.04

III Production 85.16 14.02 5.40 88.05 12.34 4.27

Yield 77.08 9.93 4.75 85.61 9.05 3.43

Area 56.86 5.25 3.45 3.74 2.74 2.69

IV Production 30.08 8.16 6.82 0.19 4.47 4.47

Yield 2.11 3.84 3.80 0.97 3.38 3.36

Area 69.35 2.00 1.11 85.19 3.19 1.23

V Production 58.8 8.31 5.33 91.55 8.83 2.57

Yield 45.74 6.87 5.06 87.57 5.79 2.04

Area 91.10 28.01 8.45 83.53 16.95 6.88

Overall Production 93.74 53.32 13.5 91.87 43.38 12.4

Yield 91.96 31.46 9.02 93.18 30.53 7.97

positive growth rates of 1.25, 3.68 and 2.40 percent respectively Abraham TP, Raheja SK (1967) An Analysis of Growth of during this period. Hence area, production and productivity of Production of Rice and Wheat Crops in India. Indian Journal of wheat have increased with highly significant growth in both Agricultural Economics 22: 2-11. Haryana and India. Trend of yield in India was similar to that of Haryana. Similar results were observed for coefficient of variation Ali MA, Singh AK (1995) Growth and Fluctuation in Area, and stability indices in production and yield of wheat in Haryana Production and Productivity of Wheat in Chattisgarh Region as well as India. of Madhya Pradesh. Indian Journal of Agricultural Economics 50: 609-614. Conflict of interests Bastine CL, Palanisami KP (1994) an analysis of growth trends in There is no conflict of interest. principal crops in Kerala. Agricultural Situation in India, 48: 885-891.

References Bhatnagar S, Saxena KK (2000) an Estimate of Area and Production of Wheat in Haryana. Agricultural Situation in India Abid S, Nisar AS, Hassan A, Farooq A (2014) Growth and trend 56: 665-667. in area, production and yield of major crops of Khyber Pakhtunkhwa, Pakistan. Asian Journal of Agriculture and Rural Bhatnagar S, Nandal, DS (1994) Growth of Wheat in Haryana. Development 4: 149-155. Agricultural Situation in India 49: 575-578.

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Ganjeer PK, Kaushik D, Lakhera ML (2017) A Review on Trend production and productivity of paddy in Bastar region of in Area, Production and Productivity of Wheat Crop in different Chhattisgarh state. Plant Archives 17: 158-160. Districts of Northern Hills of Chhattisgarh State. Bulletin of Environment, Pharmacology and Life Sciences 6: 302-304. Siju T, Kombairaju S (2001) Rice production in Tamil Nadu: A trend and decomposition analysis. Agricultural Situation India 58: 143-145. Gautam AK, Sisodia BVS (2018) Analysis of trends and growth Singh M, Supriya K (2017) Growth Rate and Trend Analysis of rate of wheat crop and forecast of its production in Uttar Pradesh. Wheat Crop in Uttar Pradesh, India. International Journal of Journal of Pharmacognosy and Phytochemistry 7: 3306-3310. Current Microbiology and Applied Sciences 6: 2295-2301.

Gupta BS, Saraswat PK (1997) Growth of rapeseed and mustard Singh, RKP, Ranjan KP (1998). Growth and instability in in Western Rajasthan. Agriculture Situation in India 54: 261-263. production of principal food grains cops: A case of backward economy. Bangladesh Journal of Agricultural Economics 21: 1-20. Kumar V, Patle GT, Singh DR, Chand R (2017) Trend of Area, Production and Productivity of Major Cereal Crops in Context of Statistical Abstract of Haryana, Department of Economics and Food Security: Sikkim, India. International Journal of Engineering Statistical Analysis, Haryana. Available on Technology Science and Research 4: 2394–3386. http://esaharyana.gov.in/en-us/State-Statistical-Abstract-of- Haryana access on 12th January, 2019. Laitonjam N, Singh R, Yumnam A, Kalai K, Meena NK (2018) Pulses Production in India: Decomposition and trend analysis. Statistical Abstract of India, Department of Economics and Plant Archives 18: 435-438. Statistical Analysis, India. Available on https://www.indiaagristat.com/agriculture-data/2/agricultural- Netam OM, Sahu LK (2017) Decade trend analysis of area, area-land-use/152/stats.aspx access on 12th January, 2019.

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Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 273 – 280

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

PARAMETERS OF AMMI MODEL TO ASSESS G×E INTERACTION OF MALT BARLEY GENOTYPES

Ajay Verma1*, V Kumar, AS Kharab, GP Singh

1ICAR-Indian Institute of Wheat & Barley Research, Karnal 132001 Haryana

Received – April 16, 2019; Revision – May 20, 2019; Accepted – June 07, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).273.280

KEYWORDS ABSTRACT

G×E interactions AMMI analysis of twenty three promising malt barley genotypes were evaluated at nine major locations of the country to interpret complex genotype by environment (GE) interactions. Combined analysis of AMMI model variance indicated the larger and highly significant G×E interaction. Seven significant IPCA’s were used ASV to calculate AMMI based measures. Type 1 measures (EV1, ASTAB1, D1), considered G12, G8, G19, G20 as desirable genotypes and G9, G2 as of unstable performance; while type 2 (EV2, ASTAB2, D2 ASTAB and ASV) considered G12, G10, G22, G15 as genotypes of choice and G9, G2 as unsuitable; type 3 (EV3, ASTAB3 and D3) pointed towards G22, G12, G23, G3 as of stable type along with G2, G9 of EV unstable type; type 5 measures (EV5, ASTAB5, SIPC5 and D5) selected G22, G12, G23, G3 genotypes and G2 & G9; type 7 utilized more than 97% of G×E interaction (EV7, ASTAB7, MASV and D D7) identified G22, G3, G12, G6 as genotypes of recommendations and G2 & G9 were detected as the MASV unstable genotypes. AMMI based measures recommended genotypes G12, G13, G22 and G3 had the moderate yield performance while G19 was of high yield. Association analysis among measures by multivariate hierarchical Ward’s clustering approach grouped into three major clusters. Largest group clubbed as many as 16 measures while yield combined with IPCA2, and SIPC1, SIPC2, SIPC3, SIPC5, SIPC7 in second group.

* Corresponding author All the articles published by Journal of Experimental Biology and Agricultural Sciences are licensed under a E-mail: [email protected] (Ajay Verma) Creative Commons Attribution-NonCommercial 4.0

International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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Parameters of AMMI model to assess GxE interaction of malt barley genotypes 274

1 Introduction AMMI model (Additive main effects and multiplicative interaction model) observed as choice of researchers with emphasis on main Barley is cultivated worldwide mainly for malt and beer production effects and interactions both (Crossa et al., 1990; Mohammadi et al., (Kilic, 2014). G×E interaction governs the identification of the stable 2015; Nowosad et al., 2018). Multi Environment trials of all crops pay yield performer genotypes suitable for a specific environment or for same attention to efficient estimation of main and interaction effects several environments (Akbarpour et al., 2014). An efficient (Kendal & Tekdal, 2016). Prime objectives of this study are application assessment of G×E interaction is very essential to determine the yield of different AMMI based measures and explore the association analysis potential of genotypes along with stable yield performance (Dehghani among different measures in identifying adapted genotypes. et al., 2010). ANOVA analysis is useful to test the significance of interaction but this test fails to partition into total variation into simple 2 Materials and Methods and cross over interactions (Flores et al., 1998). Partitioning and interpretation of G×E interaction is carried out by linear regression Twenty three promising malt barley genotypes were evaluated at nine still this technique have inherent deficiencies for confounding of main major locations of the country during cropping season of 2017-2018 effects (Tekdal & Kendal, 2018) as well as failure to account for non in field trials via randomized complete block design with four linear genotypic response (Yau, 1995). Other multiplicative model replications. Fields were prepared nicely and agronomic analysis based on principal component lacked to describe the additive recommendations were followed to harvest good crop. Parentage main effects (Sabaghnia et al., 2013; Bocianowski et al., 2019a). details and environmental conditions had mentioned in table 1 for

Table 1 Parentage details of malt barley genotypes along with environmental conditions

Code Genotype Parentage Code Environments Latitude Longitude Mean sea level G 1 DWRB181 DWRUB52/DWRB77 E 1 Bawal 28 ͦ 10 ' N 76 ͦ 59 ' E 263 G 2 RD2986 DWR39/RD-2651 E 2 Durgapura 26 ͦ 51 'N 75 ͦ 47 ' E 390 G 3 KB1638 K551/NDB1295 E 3 Hisar 29 ͦ 10 'N 75 ͦ 46 ' E 215.2 G 4 DWRB183 DWR28/DWRUB52 E 4 SG Nagar 29 o 90' N 73 ͦ 87' E 176 G 5 DWRB182 DWRUB52/DWRB78 E 5 Ludhiana 30o 54 ' N 75o 52' E 247 G 6 RD2988 DWRUB52/RD-2651 E 6 Bathinda 30 o 21 ' N 74 ͦ 95' E 208 G 7 DWBR137 DWR28/DWRUB64 E 7 Mathura 27 o 49' N 77 ͦ 67' E 187 G 8 DWRB160* DWRB62/DWRB73 E 8 Pantnagar 29 o 02 ' N 79 ͦ 48 ' E 237 G 9 DWRB184 DWRUB52/DWR81 E 9 Karnal 29 ͦ 43 ' N 76 ͦ 58 ' E 252 G 10 Andreia

G 11 PL904 DWRUB64/RD2668 G 12 RD2849 (c) DWRUB52/PL705 G 13 DWRB101 (c) DWR28/BH581 G 14 Explorer

G 15 DWRB123 (c) DWRUB54/DWR51 G 16 RD2985 Clipper/RD-2668//DWR73 G 17 BH902 (c) BH495/RD2552 G 18 KB1634 Lakhan/JB137 G 19 DWRB187 RD2035/ RD2552 G 20 ABI Voyager

G 21 Danielle

G 22 PL905 VJM 604/PL764 G 23 RD2987 Clipper/EB921//RD-2668

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275 Verma et al.

ready reference. More over grain yield was analyzed further to 3 Results and Discussion

estimate the G×E interaction component by AMMI analysis. The description of widely used measures based on AMMI analysis was The combined analysis of variance (ANOVA) showed highly mentioned for completeness. significant differences between the genotypes (G), which supported the considerable variability with respect to grain yield 푁 in this study (Table 2). Significant differences were also observed Zobel, 1994 EV1 EVF EV = 휆2 /푛 푖푛 among the environments (E). AMMI analysis of variance showed 푛=1 that environments, genotypes and G×E interaction effects 푁 accounted for 58%, 12% and 22.5% of the total variation, Sneller et al., SIPC1 SIPCF SIPC = 휆0.5 훾 1997 푛 푖푛 respectively. Variance explained by the G×E interaction effect 푛=1 was nearly two times that of genotype effects. This was further Purchase, 푆푆퐼푃퐶 1 divided into seven significant interaction principal component ASV ASV = [ (푃퐶퐼)2 + (푃퐶2)2]1/2 1997 푆푆퐼푃퐶 2 axes (IPCAs). First four IPCA’s contributed more than 80%. IPCA1 explained 31.1% of the variation affected by interaction, Annicchiaric D D = 푁 (λ 훾 )2 o, 1997 푛=1 푛 푖푛 while IPCA2, IPCA3 and IPCA4 accounted for 22.7, 16.7 and 10.6%, respectively. Since the main effect of genotypes on grain Rao & 푛 yield was highly significant, mean yield was considered as the 2 Prabhakaran, ASTAB 퐴푆푇퐴퐵 = 휆푛 훾푛푖 first parameter for assessing the yield potential of genotypes. In 2005 푛=1 this respect, G7, G9, G19, G17 and G5 had the highest and genotypes G21, G14 and G10 had the lowest average yields 푁−1 푆푆퐼푃퐶 Zali et al., 푛 2 2 across 09 environments. 푀퐴푆푉 푀퐴푆푉 = (푃퐶푛 ) + (푃퐶푛+1) 2012 푆푆퐼푃퐶푛 +1 푛=1 Large magnitude of G×E interactions for yield found in this investigation are similar to those found in other crops (Sabaghnia EV1 = EV for first IPCA; EVF = EV for seven IPCA & SIPC1 = SIPC for first IPCA, SIPC 2 = SIPC for first two IPCAs et al., 2012; Mortazavian et al., 2014). The high significance of GE interactions is indicating the studied genotypes exhibited both AMMI model computations were performed by software crossover and non-crossover types of GE interaction. Total G×E MATMODEL version 3.0 (Gauch, 2007) and SAS software version 9.3. (35116.26) was partitioned into G×E noise (3073.76) that is

Table 2 AMMI analysis of malt barley genotypes

Source df MS Level of significance % of Total SS % of GxE SS Cumulative % SS by PCA’s

Treatments 206 704.6512 *** 93.05

Genotypes 22 888.6938 *** 12.53

Environments 8 11311.33 *** 58.01

GxE 176 199.5242 *** 22.51

IPC1 29 376.2301 *** 31.07 31.07

IPC2 27 294.8774 *** 22.67 53.74

IPC3 25 234.49 *** 16.69 70.44

IPC4 23 161.5521 *** 10.58 81.02

IPC5 21 135.5105 *** 8.10 89.12

IPC6 19 89.42937 *** 4.84 93.96

IPC7 17 82.85984 *** 4.01 97.97

Residual 15 47.4968 ***

Error 621 17.46457

Total 827 188.638

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Parameters of AMMI model to assess GxE interaction of malt barley genotypes 276

8.75% and G×E signal (32042.49) of 91.25%. AMMI derived use of AMMI derived parameters based on the larger numbers of measures based on the use of significant IPCA’s were calculated IPCAs results in the most usage of G×E interaction variations. as EV1, ASTAB1, SIPC1, D1 measures (only first significant IPCA), while ASV, EV2, ASTAB2, SIPC2, D2 considered IPCA1 Minimum and maximum values of EV1 observed for G12, G8, & IPCA2 both, measures EV3, ASTAB3, SIPC3 and D3 used G20, G19 and G9, G2 while corresponding to D1 were G12, G8, three IPCAs, EV5, ASTAB5, SIPC5 & D5 (based on five IPCAs), G20, G19 and G9, G2 absolute values of ASTAB1 for G12, G8, whereas measures EV7, ASTAB7, SIPC7 and D7 utilized all G20, G19 and G9, G2 and for SIPC1 were G17, G14, G21, G6 & significant IPCAs. G9, G2, genotypes G7, G9, G19, G17 were of high yield and G21, G14 of low yield performance (Tables 3 & 4). Explained variation of G×E interaction accounted by each of IPCA exploited by defined measures, as type-1 measures Genotypes EV2 pointed towards G12, G10, G22 & G15 as benefited 31.07%, type-2 measures utilized 53.74%, type 3 desirable at the same time undesirable genotypes (G9, G2), for measures used up to 70.44%, type 5 measures benefited up to values of D2 genotypes were G12, G10, G22, G15 & G9, G2, 89.1%, while type 7 measures accounted for most of variation and whereas as per criterion of SIPC2 were G6, G14, G19, G16) utilized to the extent of benefits 97.9% (Table 2). This justifies the & G9, G4 and of ASTAB2 were G12, G10, G22, G15 & G9, G2

Table 3 Principal components analysis of malt barley genotypes

Yield IPCA 1 IPCA 2 IPCA 3 IPCA 4 IPCA 5 IPCA 6 IPCA 7 ASV MASV

G 1 DWRB181 47.94 1.2854 0.8653 -0.5628 -0.3128 1.5982 -0.1224 0.2926 1.74 3.47

G 2 RD2986 47.36 3.0039 -1.4643 2.5807 0.9765 -0.7550 -0.5527 -0.7724 3.81 6.29

G 3 KB1638 48.44 -1.4427 0.3568 0.4008 -0.7619 0.8400 0.7669 1.0419 1.73 3.03

G 4 DWRB183 49.89 1.1253 1.9575 -0.0542 -0.1159 0.5759 0.6493 -0.7277 2.36 3.63

G 5 DWRB182 52.39 1.8720 -0.0352 -2.8564 0.1709 0.6254 1.4882 -0.6779 2.19 5.68

G 6 RD2988 42.33 -1.7043 -1.7110 -1.0213 1.7796 -1.7807 0.8318 0.3624 2.63 5.57

G 7 DWBR137 58.86 -0.7372 2.0129 -0.7513 -1.2641 -0.3383 -2.1201 0.9557 2.19 5.16

G 8 DWRB160* 52.17 -0.2517 2.1653 -1.6704 0.2797 -1.8901 -0.9560 -0.5142 2.19 5.51

G 9 DWRB184 55.89 3.6077 0.2804 -0.4140 0.0493 -1.1730 -0.9440 0.0440 4.23 4.91

G 10 Andreia 42.06 0.8325 -0.4839 1.5361 -3.0298 -0.1470 0.5405 -0.2680 1.09 5.43

G 11 PL904 45.64 1.1752 1.8465 1.6417 0.7087 -0.5830 1.2382 0.8821 2.30 4.81

G 12 RD2849 (c) 49.61 0.0146 0.5695 0.3343 0.9487 0.9424 0.6381 -2.0675 0.57 3.27

G 13 DWRB101 (c) 51.64 -1.1996 -0.6031 0.5299 1.1783 1.9515 -0.4832 -0.3605 1.53 4.19

G 14 Explorer 41.22 -1.8659 -1.1477 -0.3131 -1.0421 0.2506 -0.3066 -1.5544 2.47 3.66

G 15 DWRB123 (c) 50.14 -0.6921 -0.9681 1.2982 0.0220 1.6458 -1.3177 0.1152 1.26 4.28

G 16 RD2985 47.47 -1.1497 -0.8806 0.9640 1.7789 -1.2227 -1.3075 -0.1385 1.61 4.60

G 17 BH902 (c) 54.17 -1.9871 2.1003 0.2214 0.8491 0.2077 0.3033 -0.6879 3.13 4.29

G 18 KB1634 53.83 -0.6737 1.1680 1.7332 0.7157 0.3759 0.5201 1.6729 1.41 4.07

G 19 DWRB187 55.14 0.5322 -1.3341 -2.5779 0.8436 1.2020 -0.7114 0.9174 1.47 5.40

G 20 ABI Voyager 43.25 0.3041 -3.0918 -0.2031 -1.0428 -0.2507 0.0654 0.1875 3.11 5.05

G 21 Danielle 40.83 -1.8094 0.1373 -0.0162 -2.0856 -1.3070 0.0342 -0.7561 2.12 4.44

G 22 PL905 49.08 0.6852 -0.8120 -0.7115 -0.5930 0.2742 -0.0411 1.3160 1.14 2.50

G 23 RD2987 45.97 -0.9247 -0.9280 -0.0882 -0.0529 -1.0421 1.7867 0.7368 1.43 3.70 IPCA, principal component of interaction, ASV = AMMI stability value, MASV = Modified AMMI Stability value

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Table 4 AMMI based estimates of malt barley genotypes

EV1 EV2 EV3 EV5 EV7 D1 D2 D3 D5 D7

DWRB181 0.0158 0.0121 0.0094 0.0156 0.0115 13.14 15.47 16.24 20.14 20.17

RD2986 0.0864 0.0552 0.0658 0.0447 0.0353 30.70 33.67 40.54 41.62 41.82

KB1638 0.0199 0.0107 0.0078 0.0092 0.0128 14.74 15.13 15.53 17.72 18.57

DWRB183 0.0121 0.0275 0.0184 0.0123 0.0123 11.50 21.78 21.79 22.21 22.67

DWRB182 0.0336 0.0168 0.0467 0.0296 0.0306 19.13 19.14 31.48 31.83 33.28

RD2988 0.0278 0.0303 0.0247 0.0371 0.0294 17.42 23.77 25.39 31.73 32.19

DWBR137 0.0052 0.0253 0.0193 0.0173 0.0314 7.53 20.46 21.49 23.78 27.50

DWRB160* 0.0006 0.0266 0.0299 0.0316 0.0267 2.57 20.62 25.28 28.88 29.55

DWRB184 0.1246 0.0627 0.0426 0.0307 0.0250 36.87 36.97 37.14 38.12 38.60

Andreia 0.0066 0.0046 0.0134 0.0382 0.0286 8.51 9.66 16.55 28.90 29.11

PL904 0.0132 0.0257 0.0289 0.0202 0.0227 12.01 21.18 25.59 26.53 27.78

RD2849 (c) 0.0000 0.0018 0.0017 0.0073 0.0229 0.15 5.38 6.13 11.82 13.52

DWRB101 (c) 0.0138 0.0089 0.0072 0.0231 0.0178 12.26 13.52 14.29 22.18 22.41

Explorer 0.0333 0.0240 0.0165 0.0137 0.0193 19.07 21.94 22.11 23.63 24.02

DWRB123 (c) 0.0046 0.0075 0.0124 0.0176 0.0186 7.07 11.56 16.21 20.18 21.88

RD2985 0.0127 0.0107 0.0112 0.0227 0.0222 11.75 14.40 16.69 23.48 24.93

BH902 (c) 0.0378 0.0436 0.0293 0.0201 0.0165 20.31 28.40 28.46 29.26 29.38

KB1634 0.0043 0.0098 0.0196 0.0140 0.0216 6.89 13.01 19.98 20.93 21.59

DWRB187 0.0027 0.0113 0.0365 0.0296 0.0261 5.44 13.73 26.41 28.60 29.05

ABI Voyager 0.0009 0.0540 0.0362 0.0255 0.0184 3.11 29.38 29.44 30.60 30.60

Danielle 0.0313 0.0158 0.0105 0.0270 0.0215 18.49 18.54 18.54 26.46 26.52

PL905 0.0045 0.0059 0.0062 0.0051 0.0103 7.00 10.39 12.11 13.12 13.52

RD2987 0.0082 0.0089 0.0060 0.0077 0.0186 9.45 12.89 12.92 15.00 18.97 EV = Eigenvector, AMGE = Sum across environments of GEI, SIPC = Sum of the value of the IPC Scores, D = Parameter of Annicchiarico

(Tables 4 and 5). In recent studies, agronomic concept of stability validation due to computation from first two IPCAs. Results put would be more preferred instead of static concept of stability forward by ASV measure have many similarities with the other (Karimizadeh et al., 2016). Using first two IPCAs in stability AMMI stability parameters which calculated from the first two analysis could benefits dynamic concept of stability in IPCAs scores (Carlos & Krzanowski, 2006). identification of the stable high yielder genotypes. Minimum values EV3 preferred G12, G23, G22, G13 as well of ASV recommended G12, G10, G22 & G15 as of stable unstable performance of G2, G5 while SIPC3 pointed towards G6, performance and unsuitable were G9, G2 (Table 3). Considering G19, G14, G20 and G11, G2 whereas D3 for G12, G22, G23, G13 & first two IPCAs in ASV measure used 53.7% of G×E interaction. G2, G9; ASTAB3 measure considered G12, G22, G23, G13 & G2, The two IPCAs have different values and meanings and the ASV G9 (Table 4). G22, G12, G23, G3 preferred by least values EV5 and parameter using the Pythagoras theorem and to get estimated maximum values found for G2, G10, measure SIPC5 identified values between IPCA1 and IPCA2 scores to produce a balanced G21, G6, G20, G14 and G11, G2 whereas D5 considered G12, G22, measure between the two IPCA scores (Purchase, 1997). Also, G23, G3 as suitable & G2, G9 as unsuitable ones; ASTAB5 selected ASV parameter of this investigation used advantages of cross G12, G22, G23, G3 as suitable & G2, G9 as unsuitable genotypes.

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Parameters of AMMI model to assess GxE interaction of malt barley genotypes 278

Table 5 Further AMMI based estimates for GxE interaction analysis

SIPC1 SIPC2 SIPC3 SIPC5 SIPC7 ASTAB1 ASTAB2 ASTAB3 ASTAB5 ASTAB7

DWRB181 1.29 2.15 1.59 2.87 3.04 16.89 23.96 26.74 46.15 46.77

RD2986 3.00 1.54 4.12 4.34 3.02 92.22 112.48 170.76 182.36 187.98

KB1638 -1.44 -1.09 -0.69 -0.61 1.20 21.27 22.47 23.88 33.56 44.00

DWRB183 1.13 3.08 3.03 3.49 3.41 12.94 49.15 49.18 51.70 57.65

DWRB182 1.87 1.84 -1.02 -0.22 0.59 35.82 35.83 107.22 110.30 127.34

RD2988 -1.70 -3.42 -4.44 -4.44 -3.24 29.69 57.35 66.48 114.36 119.61

DWBR137 -0.74 1.28 0.52 -1.08 -2.24 5.55 43.84 48.78 62.10 96.55

DWRB160* -0.25 1.91 0.24 -1.37 -2.84 0.65 44.95 69.37 96.06 103.54

DWRB184 3.61 3.89 3.47 2.35 1.45 133.02 133.76 135.26 145.33 151.06

Andreia 0.83 0.35 1.88 -1.29 -1.02 7.08 9.30 29.94 101.80 104.11

PL904 1.18 3.02 4.66 4.79 6.91 14.11 46.33 69.92 76.32 90.93

RD2849 (c) 0.01 0.58 0.92 2.81 1.38 0.00 3.07 4.04 17.56 46.37

DWRB101 (c) -1.20 -1.80 -1.27 1.86 1.01 14.71 18.15 20.60 59.25 61.54

Explorer -1.87 -3.01 -3.33 -4.12 -5.98 35.58 48.03 48.88 57.82 73.24

DWRB123 (c) -0.69 -1.66 -0.36 1.31 0.10 4.90 13.75 28.50 48.28 59.51

RD2985 -1.15 -2.03 -1.07 -0.51 -1.96 13.51 20.84 28.97 64.60 75.69

BH902 (c) -1.99 0.11 0.33 1.39 1.01 40.35 82.04 82.47 88.41 91.91

KB1634 -0.67 0.49 2.23 3.32 5.51 4.64 17.53 43.82 48.85 67.74

DWRB187 0.53 -0.80 -3.38 -1.33 -1.13 2.89 19.71 77.86 93.97 102.37

ABI Voyager 0.30 -2.79 -2.99 -4.28 -4.03 0.95 91.28 91.64 100.59 100.83

Danielle -1.81 -1.67 -1.69 -5.08 -5.80 33.46 33.64 33.64 80.08 83.59

PL905 0.69 -0.13 -0.84 -1.16 0.12 4.80 11.03 15.46 18.75 29.38

RD2987 -0.92 -1.85 -1.94 -3.04 -0.51 8.74 16.88 16.95 24.90 48.72 SIPC1 = SIPC for first IPCA, SIPC 2 = SIPC for first two IPCAs, … for

According to D7 minimum values G12, G22, G3, and G23 G9, G2 as unsuitable; as per type 3 (EV3, ASTAB3 and D3), G22, were genotypes of stable yield while G2 and G9 as G12, G23, G3 were stable genotypes and G2, G9 of unstable type; undesirable; SIPC7 observed G14, G21, G20, G6 as of stable according to the type 5 of AMMI parameters (EV5, ASTAB5, & G11, G18 of unstable yield (Tables 4 and 5). EV7 pointed SIPC5 and D5), genotypes G22, G12, G23, G3 and G2 & G9; towards G22, G1, G5, G4 & G2, G7. Measure ASTAB7 lastly based on the type 7 (EV7, ASTAB7, MASV and D7), identified G22, G3, G12, G1 as desirable and G2, G9 for genotypes G22, G3, G12, G6 and G2 & G9 were detected as the unstable behavior over the studied environments. Composite unstable genotypes. Considering all of the AMMI based measures, measure MASV selected G22, G3, G12, G1 as of stable only genotypes G12, G13, G22 and G3 had the moderate yield performance and G2, G5 not recommended for cultivation due performance while G19 was of high yield. to unstable yield behavior. To better understand the relationship among the AMMI based Finally type 1 of AMMI measures (EV1, ASTAB1, D1), estimates along with yield, principal component analysis (PCA) considered G12, G8, G19, G20 as desirable genotypes and G9, G2 was performed. The relationship among these estimates is as of unstable performance; based on the type 2 (EV2, ASTAB2, graphically displayed in a plot of PC1 versus PC2. AMMI based D2 and ASV) G12, G10, G22, G15 were genotypes of choice and measures along with yield could be divided into three major

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279 Verma et al.

0.7

SIPC5 0.6 SIPC7 SIPC3 SIPC2 0.5

IPCA0.4 2 IPCASIPC1 1 Mean 0.3 IPCA 5

0.2 IPCA 3

IPCA 4 IPCA 7 0.1 EV1ASTAB1 IPCA 6 D1 0.0 EV3ASTAB3 ASTAB2 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 EV2 0.5D3 0.6 D2ASV

-0.1 ASTAB7 D5ASTAB5D7 EV7 -0.2 MASV EV5

-0.3

Figure 1 Biplot representation of AMMI based estimates clusters (Figure 1). Largest group I clubbed 16 measures as ASV, Bocianowski J, Warzecha T, Nowosad K, Bathelt R (2019a) MASV, D1, D2, D3, D5, D7, with EV1, EV2, EV3, EV5, EV7, Genotype by environment interaction using AMMI model and as well as ASTAB2, ASTAB3 ASTAB1, ASTAB7. Group II estimation of additive and epistasis gene effects for 1000-kernel contains yield with IPCA2, and SIPC1, SIPC2, SIPC3, SIPC5, weight in spring barley (Hordeum vulgare L.) Journal of Applied SIPC7, whereas IPCA5 was lying far way. Group III contains Genetics 60:127–135 IPCA3, IPCA4, IPCA6, IPCA7 whereas SIPC1 and IPCA1 were observed as outliers. Bocianowski J, Niemann J, Nowosad K (2019b) Genotype-by environment interaction for seed quality traits in inter specific Each of the AMMI stability parameters relates to a different cross-derived Brassica lines using additive main effects and concept of yield stability and may be useful to plant breeders multiplicative interaction model. Euphytica 215:7 attempting to select genotypes with high, stable and predictable yield across environments (Mohammadi et al., 2015). However, it Carlos T dos SD, Krzanowski WJ (2006) Choosing Components In seems that there is no need to consider all of these measures The Additive Main Effect and Multiplicative Interaction (AMMI) simultaneously, and a few of them should be used in as per models. Scientia Agricola (Piracicaba, Braz.), 63:169-175. maximum usage of G×E interaction sum of squares. AMMI Crossa J, Gauch HG, Zobel RW (1990) Additive main effects and analysis has been observed as useful for exploring complex G×E multiplicative interaction analysis of two international maize interaction, improving selections and increasing experimental cultivar trials. Crop Science 30: 493–500. efficiency (Sabaghnia et al 2013; Bocianowski et al., 2019b).

Conflict of interests Dehghani H, Sabaghpour SH, Ebadi A (2010) Study of genotype × environment interaction for chickpea yield in Iran. Agronomy There is no conflict of interest. Journal 102:1–8.

References Flores F, Moreno MT, Cubero JI (1998)A comparison of univariate and multivariate methods to analyze environments. Akbarpour O, Dehghani H, Sorkhi B, Guach G (2014) Evaluation of Field Crops Research 56: 271–286 Genotype × Environment Interaction in Barley (Hordeum Vulgare L.) Based on AMMI model Using Developed SAS Program. Gauch HG (2007) MATMODEL version 3.0: Open source Journal of Agricultural Science & Technology 16: 919-930. software for AMMI and related analyses. Crop and Soil Sciences, Cornell University, Ithaca, NY Annicchiarico P (1997) Joint regression vs AMMI analysis of genotype × environment interactions for cereals in Italy. Karimizadeh R, Ali A, Chinipardaz R, Sofalian O, Ghaffari A Euphytica 94:53–62. (2016) Determining Yield Stability And Model Selection By

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Parameters of AMMI model to assess GxE interaction of malt barley genotypes 280

AMMI Method In Rain-Fed Durum Wheat Genotypes. Turkish Rao AR, Prabhakaran VT (2005) Use of AMMI in simultaneous Journal of Field Crops 21: 174-183 selection of genotypes for yield and stability. Journal of the Indian Society of Agricultural Statistics 59:76-82. Kendal E, Tekdal S (2016) Application of AMMI Model for Evolution Spring Barley Genotypes in Multi-Environment Trials. Sabaghnia N, Mohammadi M, Karimizadeh R(2012) The Bangladesh Journal of Botany 45: 613-620. Evaluation of Genotype × Environment Interactions of Durum Wheat’s Yield Using of the AMMI Model. Agriculture & Forestry Kilic H (2014) Additive Main Effect and Multiplicative 55: 5–21. Interactions (AMMI) Analysis of Grain Yield in Barley Genotypes across Environments. Journal of Agricultural Sciences Sabaghnia N, Mohammadi M, Karimizadeh R (2013) Parameters 20: 337-344. of AMMI model for yield stability analysis in durum wheat. Agriculturae Conspectus Scientificus 78 : 119-124. Mohammadi M, Sharifi P, Karimizadeh R, Jafarby JA, Khanzadeh H, Hosseinpour T, Poursiabidi MM, Roustaii M, Hassanpour HM, Sneller CH, Kilgore-Norquest L, Dombek D (1997) Repeatability Mohammadi P (2015) Stability of grain yield of durum wheat of yield stability statistics in soybean. Crop Science 37: 383-390. genotypes by AMMI model. Agriculture & Forestry 61: 181-193. Tekdal S, Kendal E (2018) AMMI Model to Assess Durum Mortazavian SMM, Nikkhah HR, Hassani FA, Sharif-al-Hosseini Wheat Genotypes in Multi-Environment Trials. Journal of M, Taheri M, Mahlooji M (2014) GGE Biplot and AMMI Agricultural Science & Technology 20: 153-166 Analysis of Yield Performance of Barley Genotypes across Different Environments in Iran. Journal of Agricultural Science & Yau SK (1995) Regression and AMMI analysis of genotype × Technology 16: 609-622. environment interactions: An empirical comparison. Agronomy Journal 87: 121–126. Nowosad K, Tratwal A, Bocianowski J (2018) Genotype by environment interaction for grain yield in spring barley using Zali H, Farshadfar E, Sabaghpour SH, Karimizadeh R (2012) additive main effects and multiplicative interaction model. Cereal Evaluation of genotype × environment interaction in chickpea Research Communications 46:729–738 using measures of stability from AMMI model. Annals of Biological Research 3: 3126-3136. Purchase JL (1997) Parametric analysis to describe G × E interaction and yield stability in winter wheat. Ph.D. thesis Zobel R (1994) Stress resistance and root systems. In Proceedings submitted to the Department of Agronomy, Faculty of of the Workshop on Adaptation of Plants to Serious Stresses. 1–4 Agriculture, University of the Orange Free State, Bloemfontein, August. INTSORMIL Publication 94-2, Institute of Agriculture South Africa. and Natural Recourses. Lincoln, USA: University of Nebraska.

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Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 281 – 288

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

GROWING CONDITION INFLUENCES VARIATION IN INITIATION TO WILTING OF FLOWERS OF WINTER ANNUALS

Bikash Bhattarai*1, Soumen Maitra2, Rocky Thokchom3

1Research Scholar, Department of Horticulture, Sikkim University, 6th Mile Samdur, Tadong, Gangtok - 737102, Sikkim, India 2Department of Floriculture, Medicinal and Aromatic Plants, Faculty of Horticulture, Uttar Banga Krishi Viswavidyalaya, Pundibari, Cooch Behar - 736165, West Bengal, India 3Department of Floriculture, School of Horticulture, Pandit DeenDayal Upadhyay Institute of Agricultural Sciences, Utlou, Bishnupur- 795134, Manipur, India.

Received – April 16, 2019; Revision – May 28, 2019; Accepted – June 07, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).281.288

KEYWORDS ABSTRACT

Ornamental annuals Present study was conducted to study the differences in flowering behavior of twenty different ornamental winter annuals, mostly used for landscaping purpose under different agro-climatic Winter annuals conditions, in the Terai region of West Bengal, India under open field and protected condition. Flowering period Flowering parameters such as Days required for flower bud initiation (FBI), Days required for flower bud development (FBD), Days required for blooming, Days required for wilting of flower were recorded in each plot and their average was calculated. Calendula officinalis showed earliness in FBI (23.25 DAT), FBD (32.88 DAT), blooming (35.50 DAT) and wilting (43.00 DAT) under open condition and the late flowering was found in Antirrhinum majus (74.67 DAT), whereas, the delayed wilting was observed in Helichrysum bracteatum (94.30 DAT) both under protected condition. Iberis umbellata required minimum time period (6.17 days) to reach flower bud development from flower bud initiation stage. The shortest period for wilting of flowers was recorded in Eschscholtzia californica (2.00 days) under protected condition, while, Helichrysum bracteatum required the maximum time period (21. 84 days) from blooming to wilting.

* Corresponding author All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Bikash Bhattarai) Creative Commons Attribution-NonCommercial 4.0

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Growing condition influences variation in initiation to wilting of flowers of winter annuals 282

1 Introduction both under open field and protected conditions to develop a database on the performance of ornamental annuals in the Terai An annual plant is a plant that completes its life cycle within a region of West Bengal for more diversified use of ornamental particular growing season of the year. Whereas, annual plant which annuals in landscape, garden display as well as in commercial having short period of lifespan is known as flowering annuals. floriculture. Plants which are sown during winter months starting from October to first week of November and flowering commences within 2 Materials and Methods February are called as winter flowering annuals or winter annuals for the plains. (Randhawa & Mukhopadhyay, 1986) Plants with Present experiment was carried out at the instructional field of strong vernalization requirement are often referred as winter Department of Floriculture, Medicinal and Aromatic plants, annuals. Winter flowering annuals provides excellent relaxation, Faculty of Horticulture, Uttar Banga Krishi Viswavidyalaya, attracts people when planting in a mass and serve as beautification Pundibari, Cooch Behar, West Bengal, India. Experiment was in any landscape plan (Brown, 2012). Annuals are also used as designed in Randomized Block Design with 20 different winter bedding plants, rockery, window baskets, garden plants and flowering annuals, each annual were replicated twice (4 plants per herbaceous borders in garden. each replication). The detail of the selected flowering species is depicted in Table 1. Seeds of 20 winter flowering annuals were Flowering in plant is an inductive process which includes initiation of floral meristem in which the apical meristem modified towards Table 1 Details of the winter flowering ornamental annuals used floral development (McDaniel et al., 1992). Murfet (1977) Sl. English common Botanical name Plant family summarized as the flowering is the end result of physiological No. name processes, biochemical sequences, and gene action, which are 1 Snapdragon Antirrhinum majus L. Scrophulariaceae influenced by environmental stimuli and the passage of time (Munir Pot Marigold / 2 Calendula officinalis L. Compositae 2003; Zheng et al., 2006) as well as genotype; a genotype can take Calendula Helichrysum two seasons to flower in an environment whereas it may flower Straw Flower / 3 bracteatum (Vent.) Compositae within a single season in different environment. Usually, after Helichrysum Andrews completing of juvenile and developmental phases plants go through 4 Petunia Petunia hybridaVilm Solanaceae reproductive phase responding to the environmental factors Livingstone Daisy / Mesembryanthemum (temperature, photoperiod). The literatures related to the winter 5 Aizoaceae Mesembryanthemum criniflorum L. annuals like effects of differential temperature on the growth, 6 Larkspur Delphinium ajacis L. Ranunculaceae morphology and flowering of Antirrhinum majus, flower production of calendula, growth and flowering of Californian poppy, 7 Daisy / English Daisy Bellis perennis L. Compositae Eschscholtzia Helichrysum bracteatum, Coreopsis, Cornflower, Sweet Williams 8 Californian Poppy Papaveraceae (Krogt, 1980; Kanamadi et al., 1999; Kazinczi et al., 1999; Shang et californica Cham. al., 2003; Mili & Sable, 2003; Singh, 2005; Dhatt & Kumar, 2007; 9 Shirley Poppy Papaver rhoeas L. Papaveraceae Ibrahim et al., 2010), flower initiation and development of Petunia 10 Lupin Lupinus hartwegii L. Leguminosae and Viola (Mattson & Erwin, 2003), responds of Calendula, 11 Sweet Pea Lathyrus odoratus L. Leguminosae Chrysanthemum, Pansy and Snapdragon to high temperatures, Brachycome / Brachycome iberidifolia 12 Compositae impact of reduce temperature and irradiance flowering and growth Swan River Daisy Benth. of four annual bedding plants (Warner & Erwin, 2006; Boldt & Dimorphotheca / Dimorphotheca 13 African Daisy / Compositae Atland, 2019) and assessment of height, earliness and biomass aurantiaca DC. production in winter annuals (Bhattarai et al., 2019) revealed the Cape Marigold Phlox drummondii 14 Phlox Polemoniaceae variation on plant height, growth habit, shape, size, color of flowers, Hook. time and duration of flowering within the genotypes, even though 15 Sweet William Dianthus barbatus L. Caryophyllaceae these are the species of same growing season. But reports regarding Coreopsis / Calliopsis 16 Coreopsis tinctoria L. Compositae the comparative study and the performance of diverse winter / Tick-seed ornamental annuals under different growing environments are in Dianthus / 17 Dianthus chinensis L. Caryophyllaceae scarcity. Winter annuals being a source of garden decoration are Common Pink need precise documentation and characterization to satisfy the 18 Candytuft Iberis umbellata L. Cruciferae gardening practices and landscaping principles. In this study, an 19 Cornflower Centaurea cyanus L. Compositae attempts have been made to study the comparative flowering 20 Pansy Viola tricolor L. Violaceae behavior of twenty different winter flowering ornamental annuals

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283 Bhattarai et al. sown on October 2012 in the seed bed. Beds were prepared Table 2 Spacing of the selected Genotypes having a dimension of 3m x 1m and height of the beds was Spacing Genotypes maintained up to 15 cm from the ground level. 10 g seeds of each winter flowering annual (10x20=200g) were sown in 5 seed beds, Daisy, Phlox, Dianthus, Californian Poppy, seeds of 4 winter annuals were sown in each bed. The initial 25cm × 25cm Petunia, Shirley Poppy, Coreopsis, Sweet nutrient provided was 5 kg FYM along with 10 g each of N2, P2O5 2 -1 and K2O / m . Copper oxychloride at 2 gL of water was sprayed William, Pansy, Candytuft as preventive measure in the seed bed twice at 10 and 20 days after the seedling emergence. Brachycome, Ice Plant, Antirrhinum, Lupin, 30cm × 30 cm Seedlings of 28 days were transplanted in the main field with Cornflower, Larkspur, Helichrysum spacing varies with the genotypes (Table 2). Repeated ploughing was followed to bring land to make fine tilth. The entire 50cm × 30 cm Calendula, Dimorphotheca, Sweet Pea experimental land was divided into raised beds measuring 1.0 m ×

1.0 m and there were 40 plots in each open field and polyhouse condition. Each bed was separated to the other through a 30 cm wide path in both ways. Winter flowering annuals (20 Nos.) were a Open Field planted in a same manner in open field as well as in polyhouse 40 Temperature condition. For present experiment, the glavanized iron pipe frame 35 Max polyhouses having 200 gauges UV stabilized polyethylene sheet 30 Open Field as cladding material and sides were covered with insect proof 25 Temperature white nets with side vents open facility were used (zero energy Min polyhouse). 20 15 Polyhouse Temperature The average minimum and maximum temperature of open and 10 0 0 Max protected condition varied from 7.96 C and 11.31 C during 5 January to 30.380C and 34.740C during October respectively. The Polyhouse 0 relative humidity of the study area varies from 41.87 to 98.03%. Temperature Oct Nov Dec Jan Feb Mar Apr Min Meteorological data for open field was obtained from Gramin Krishi Mausam Seva, AMFU-Pundibari, Coochbehar, West Bengal, India and polyhouse temperature and relative humidity were measured by digital hygrometer.Consequently, the area is warm and humid except a short spell of winter extending from December to February. The meteorological data of the study b Open Field period is given in Figure 1. 120 Relative Humidity Max 2.1 Parameters recorded: 100

Flowering parameters such as days required for flower bud initiation 80 Open Field Relative Humidity (FBI), days required for flower bud development (FBD), Days 60 Min required for blooming, Days required for wilting of flower were recorded in each plot and their average was calculated. Data were 40 Polyhouse analyzed using GLM procedure of statistical system (SAS) Software Relative Humidity 20 (Version 9.3). Design of the experiment was based on randomized Max block design with two replications in both the condition and 0

parameters were tested at the 5% level of significance. Polyhouse

Jan

Oct

Apr

Feb

Dec Nov

Mar Relative Humidity 3 Results and Discussion Min

Twenty different winter flowering ornamental annuals when Figure 1 Monthly mean meteorological data of open field and grown under open and protected conditions; the flowering polyhouse during the period of experiment a) Temperature and b) commences earlier in open field and later in polyhouse situation. Relative Humidity.

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Growing condition influences variation in initiation to wilting of flowers of winter annuals 284

Table 3 Days required for flower bud initiation (FBI), flower bud development (FBD), blooming and wilting of winter annuals in open field and in polyhouse condition.

Open field Polyhouse Genotypes FBI FBD Blooming Wilting FBI FBD Blooming Wilting (DAT) (DAT) (DAT) (DAT) (DAT) (DAT) (DAT) (DAT) Daisy 40.00 46.70 48.90 58.40 60.88 68.88 72.63 80.00

Phlox 43.80 53.50 55.80 59.30 54.30 65.10 67.80 71.10

Ice Plant 39.34 51.75 55.75 72.00 62.84 74.50 77.17 87.84

Helichrysum 38.67 55.00 60.33 82.17 53.84 69.10 75.67 94.30

Larkspur 48.75 64.88 67.13 79.75 55.17 71.17 73.67 84.34

Antirrhinum 47.00 57.17 60.50 67.33 74.67 86.17 89.00 93.17

Calendula 23.25 32.88 35.50 43.00 29.34 39.67 42.17 49.34

Sweet William 42.34 54.17 56.50 62.67 40.00 50.00 52.50 60.67

Petunia 47.70 55.50 57.50 61.40 67.00 75.13 77.50 83.25

Lupin 51.03 59.43 61.50 65.98 36.75 44.50 46.00 52.25

Brachycome 45.25 55.75 58.33 62.92 62.25 70.50 73.00 77.25

Cornflower 30.82 50.42 52.97 60.10 49.25 65.88 68.50 75.50

Dimorphotheca 40.60 55.20 58.00 65.80 59.00 67.92 71.21 79.84

Dianthus 45.63 55.88 58.38 64.50 48.88 58.50 61.50 69.50

Californian Poppy 45.67 53.75 55.84 59.00 65.75 75.00 77.00 79.00

Shirley Poppy 44.38 55.38 57.13 59.75 52.17 63.34 65.17 68.00

Pansy 26.23 34.15 36.35 46.13 56.94 65.10 67.47 76.77

Candytuft 31.50 39.34 41.17 48.33 34.34 40.50 42.17 50.00

Coreopsis 34.46 51.09 53.25 60.59 44.45 63.73 65.85 72.80

Sweet Pea 37.17 46.84 49.50 56.17 38.25 47.00 49.50 58.75

Pr>F <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001

SEM 1.27 1.71 1.80 1.89 2.51 2.38 2.38 2.59

CD at 5% 3.77 5.07 5.32 5.60 7.44 6.99 7.04 7.68 *DAT – Days After Transplanting

Results of study revealed that Calendula reached the flower bud open field, from FBD to blooming in Helichrysum (6.57 days) initiation stage earliest (23.25 DAT) and showed resultant earlier under polyhouse and from blooming to wilting in Helichrysum flower bud development (32.88 DAT) and full blooming (35.50 (21. 84 days) in open field condition (Table3). DAT) as well as wilting of flowers (43.00 DAT) under open field condition. Delayed flowering was observed in Antirrhinum (74.67 Annuals showed marked variation to reach into their reproductive DAT) while delayed wilting was observed with Helichrysum phase classifying them as early flowering, mid-season flowering (94.30 DAT) under polyhouse situation. Least time period or late bloomers. However, this is well established since time required to reach FBD from FBI was observed in candytuft (6.17 immemorial but the quantitative measurements of these durations days), blooming from FBD in Lupin (1.50 days) and wilting of were lacking. Moreover, the changes in duration under varied flowers from blooming was recorded in Californian poppy (2.00 growing conditions also represent the physiological changes days) under polyhouse, whereas, highest time period required within the species as an influence of the growing environment. In from FBI to FBD was noticed in Cornflower (19.60 days) under this experiment all the winter ornamental annual species except

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285 Bhattarai et al.

Table 4 Grouping of genotypes on the basis of FBI and FBI to FBD

FBI FBI TO FBD Means with the same letter are not significantly different Means with the same letter are not significantly different Open field condition Polyhouse condition Open field condition Polyhouse condition ‘t’ grouping Genotypes ‘t’ grouping Genotypes ‘t’ grouping Genotypes ‘t’ grouping Genotypes A Lupin A Antirrhinum A Cornflower A Coreopsis B A Larkspur B Petunia B A Coreopsis B A Cornflower B A C Petunia C B Californian Poppy B A Helichrysum B Larkspur B D C Antirrhinum C B D Ice Plant B A Larkspur B Helichrysum B E D C Californian Poppy C E B D Brachycome B C Dimorphotheca C Ice Plant B E D C Dianthus F C E B D Daisy D C Sweet William D C Antirrhinum B E D C Brachyeome F C E G D Dimorphotheca D C E Ice Plant D C E Shirley Poppy E D C Shirley Poppy F E G D Pansy D E Shirley Poppy D C E Phlox F E D Phlox F H E G Larkspur D E Antirrhinum D C E Calendula F E G Sweet William F H G Phlox D E Brachycome D C E Sweet William F G H Dimorphotheca F H G Helichrysum D E Dianthus D C E Dianthus G H Daisy H G Shirley Poppy D F E Phlox D F C E Californian Poppy G H Ice Plant H I Cornflower D F E Sweet Pea D F C E Dimorphotheca G H Helichrysum H I Dianthus D F E Calendula D F C E Sweet Pea I H Sweet Pea J I Coreopsis D F E Lupin D F E Brachycome J I Coreopsis J K Sweet William F E Californian Poppy F E Pansy J Candytuft J K Sweet Pea F E Pansy F E Petunia J Cornflower L K Lupin F E Candytuft F E Lupin K Pansy L K Candytuft F E Petunia F E Daisy K Calendula L Calendula F Daisy F Candytuft

Sweet William and Lupin takes higher time period to reach the agro-climatic and microclimatic condition. However, only 8 FBI under polyhouse cultivation as compared to the open field winter annuals (Ice plant, Antirrhinum, Calendula, Lupin, condition. As compared to both the cases Calendula showed the Brachycome, Californian Poppy, Coreopsis and Sweet Pea) took earliest FBI whereas the delayed effect was found variable as in maximum time period to reach the blooming stage under open open field condition, Lupin reached the FBI stage last of all (51.03 field condition than polyhouse. Data presented in Table 5, on the DAT) and in polyhouse condition Antirrhinum reached the FBI basis of growth of flower buds showed 8 classes under protected stage last of all (74.67 DAT). Findings of present study are in condition as compared to 7 classes under open field situation close conformity with Cavins et al. (2000), the author reported reflecting wider heterogeneity of flower bud maturity by the that warmer greenhouses in each year extended production time genotypes under polyhouse situation might be due to differential for Antirrhinum. response to microclimate. It was found that the in-situ longevity of flowers under open field was found better than polyhouse Based on the results through the ‘t’ grouping of LSD means, the condition except Sweet Pea, Candytuft, Shirley poppy, genotypes reflected 12 classes of data under polyhouse condition Dimorphotheca, Lupin, Petunia and Sweet William. Grouping of as compared to 10 under open field (Table 4) showed more genotypes showed marked variation within the genotypes and it is heterogeneity of growth of winter annuals under protected also become useful to select the relative placement of annuals in condition. Groping of genotypes resulted ten winter annuals out of several garden components, herbaceous borders and in outdoor twenty (Helichrysum, Larkspur, Sweet William, Lupin, landscaping. Brachycome, Cornflower, Dimorphotheca, Dianthus, Candytuft and Sweet Pea) required higher time period to attain FBD from In any landscaping condition synchronized blooming is an FBI under open field as compared to polyhouse. Although, data essential principle to use its best effect along with color pertaining from the table 4 showed that in both the cases combination of winter annuals (Lindgren et al., 2007; Love et al., genotypes were grouped into 6 (Table 4) but wide variation in the 2009). The current experiment gave the precise database of the growth of flower bud was also noticed as the same species were comparative performance of twenty different winter annuals with not grouped in a similar pattern showing differential response to respects to the duration between the major stages of flowering in

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Growing condition influences variation in initiation to wilting of flowers of winter annuals 286

Table 5 Grouping of genotypes on the basis of FBD to Blooming FBD to Blooming Means with the same letter are not significantly different Open field condition Polyhouse condition ‘t’ grouping Genotypes ‘t’ grouping Genotypes A Helichrysum A Helichrysum B Ice Plant B Daisy C B Antirrhinum C B Dimorphotheca C D Dimorphotheca C B D Dianthus C D E Sweet Pea C E D Antirrhinum C D E Calendula C F E D Phlox D E Brachycome C F E D Ice Plant F D E Cornflower C F E D Cornflower F D E Dianthus G C F E D Calendula F G D E Sweet William G C F E D Larkspur F G D E Phlox G C F E D Brachycome F G D E Larkspur G C F E D Sweet William F G D E Daisy G C F E D Sweet Pea F G D E Pansy G H F E D Petunia F G D E Coreopsis G H F E D Pansy F G D E Californian Poppy G H F E Californian Poppy F G E Lupin G H F Shirley Poppy F G E Petunia G H Candytuft F G Candytuft G H Coreopsis G Shirley Poppy H Lupin

both ways from the day after transplanting as well as from the Conclusion preceding stage. This experiment was performed to provide the information about winter annuals for the growers to sow The results obtained from the present experiment revealed that the the late bloomers well ahead with a quantitative measurement. variability exists within the genotypes under each growing Various researchers also found that the species-wise variation environment or in the same genotype under different growing regarding different durations might be due to the combination conditions. To exert the best effect in any landscaping situation, of several factors like environmental stimuli, inherent growth especially in herbaceous borders - simultaneous blooming is an pattern, soil condition, genotypic configuration of the species essential principle with color combination. To achieve this (Munir et al., 2004; Inaba & Ohshiro, 2005; Ibrahim et al., objective, it is essential to categorize the plants accordingly by 2010; Ali, 2013). Apart from that, the variation in maturity comparing its flowering habit and colour combination. was observed due to the effect of temperature under different Conflict of Interest growing conditions during flowering period (Niu et al., 2000; Kang & Iersel, 2001; Niu et al., 2006; Warner, 2010). No conflict of interest. Fluctuation in flowering behavior under open and protected condition was observed in this experiment due to temperature References and light quality was corroborated by the observations of Kumar & Kaur (2000) and Warner & Erwin (2002) also Ali MA (2013) Effect of different concentrations (NPK) on noticed significant variation in FBI and other phases of flower growth and flowering of Helichrysum bracteatum. International development within the different ornamental annual species. Journal of Agriculture and Crop Sciences5: 1966-1968.

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287 Bhattarai et al.

Bhattarai B, Maitra S, Thokchom R (2019) Assessment of Height, Nebraska Landscape, I. Flowering plants. University of Nebraska- Earliness and Biomass Production in Selected Winter Flowering Lincoln Extension. http://ianrpubs.unl.edu/live/g1774/build/g1774.pdf. Ornamental Annuals for Better Utilization in Landscaping. The Pharma Innovation 8: 59-64 Love SL, Noble K, Parkinson S, Bell S (2009) Herbaceous ornamentals: annuals, perennials and ornamental grasses. Boldt JK, Atland JE (2019) Timing of short -term reduction Short-Season, high altitude gardening 1-16. in temperature and irradiance affects growth and flowering http://www.cals.uidaho.edu/edcomm/pdf/bul/bul0861.pdf. of four annual bedding plants. Horticulturae 5: 15 https://doi.org/10.3390/horticulturae5010015. Mattson NS, Erwin JE (2003) Temperature affects flower initiation and development rate of Impatiens, Petunia, and Viola. Brown SH (2012) Colorful plant beds for South Florida and Acta Horticulturae 624: 191-197. similar climates. Lee County Extension, Fort Myers, Florida 239: 533-7513. McDaniel CN, Singer SR, Smith SME (1992) Developmental states associated with the floral transition. Developmental Biology Cavins TJ, Dole JM, Stamback V (2000) Unheated and minimally 153: 59-69. heated winter greenhouse production of specialty cut flower. Horticulture Technology 10: 793-799. Mili R, Sable AS (2003) Effect of planting density and nitrogen levels on growth and flower production of Calendula (Calendula Dhatt KK, Kumar R(2007) Effect of planting time and spacing on officinalis L.). Indian Journal of Horticulture 60: 399-403. plant growth, flowering and seed yield in Coreopsis lanceolataand Coreopsis tinctoria. Journal of Ornamental Horticulture10: 105-109. Munir M (2003) A study on the effects of environmental factors affecting the phases of flower development in Antirrhinum majus Ibrahim SMM, Taha LS, Farahat MM (2010) Influence of foliar L..Ph.D. Thesis submitted to the Department of Horticulture and application of Peptone on growth, flowering and chemical composition Landscape, School of Plant Sciences, The University of Reading, ofHelichrysum bracteatumplants under different irrigation United Kingdom. intervals. Ozean Journal of Applied Sciences 3: 143-155. Munir M, Jamil M, Baloch JUD, Khattak KR (2004) Growth and Inaba Z, Ohshiro M (2005) Effects of planting density and flowering ofAntirrhinum majus L. under varying temperatures. methods of raising seedlings on flowering, yield and quality of cut International Journal of Agriculture and Biology 6: 173-178. flowers in snapdragons (Antirrhinum majus L.). Environment Control in Biology 43: 201-210. Murfet IC (1977) Environmental interaction and genetics of flowering. Annual Review of Plant Physiology 28: 253-278. Kanamadi VC, Dharmatti PR, Patil S, Gangadharappa PM, Patil BC (1999) Propagation of straw flower (Helichrysum bracteatum Niu G H, Heins RD, Cameron AC, Carlson WH (2000) Day and night Andr.) by stem cuttings. Advances in Plant Science Research in temperatures, daily light integral and CO2 enrichment affect growth India 9:113-115. and flower development of pansy (Viola x wittrockiana). Journal of the American Society for Horticultural Science 125: 436-441. Kang JG, Iersel MWV (2001) Interactions between temperature and fertilizer concentration affect growth of subirrigated petunias. Niu GH, Rodriguez DS, Wang YT (2006) Impact of drought and Journal of Plant Nutrition 24: 753-765. temperature on growth and leaf gas exchange of six bedding plant species under greenhouse conditions. Hort Science 41: 1408-1411. Kazinczi G, Hunyadi K, Lukacs D (1999) Data on the biology of Cornflower (Centaurea cyanus L.) I. Germination biology and Randhawa GS, Mukhopadhyay A (1986) Floriculture in India. growth analysis. Novenyvedelem 35: 45-52. Allied Publishers Private Limited, 58-60.

Krogt TVD (1980) The flowering of Sweet Williams in the Shang H, Deitzer GF, Cox LJD (2003) Differential temperature winter. Vakbladvoor de Bloemisterij 35: 34-35. (DIF) effects on the growth, morphology and flowering of Antirrhinum majus L. (Snapdragon) cultivars. Acta Horticulturae Kumar R, Kaur K (2000) Effect of planting time and cultivars on 624: 177-183. growth, flowering and seed yield in Phlox (Phlox drummondii). Seed Research 28: 23-26. Singh AK (2005) Growth and seed yield in California poppy (Eschscholtzia californica Chamisso) as influenced by plant Lindgren DT, Streich AN, Todd KA, Rodie SN(2007) Annuals for growth regulators. Journal of Ornamental Horticulture 8: 159-160.

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Warner RM (2010) Temperature and photoperiod influence Warner RM, Erwin JE (2006) Prolonged high temperature flowering and morphology of four Petunia spp. HortScience 45: exposure differentially reduces growth and flowering of 12 Viola 365-368. x wittrockiana Gams. cvs. ScientiaHorticulturae108: 295-302.

Warner RM, Erwin JE (2002) Photosynthetic responses of heat- Zheng ZL, Yang Z, Jang JC, Metzger JD (2006) Phytochromes tolerant and heat-sensitive cultivars of Impatiens hawkeri and A1 and B1 have distinct functions in the photoperiodic control of Viola x wittrockiana to high temperature exposures. Acta flowering in the obligate long day plant Nicotiana sylvestris. Plant Horticulturae 580: 215-219. Cell & Environment 29: 1673-1685.

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Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 289 – 294

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

INFLUENCE OF ENVIRONMENTAL FACTORS ON THE POPULATION DYNAMICS OF CHILLI THRIPS, Scirtothrips dorsalis (HOOD) AND APHID, Aphis gossypii (GLOVER)

Deepak Kumar, Kamal Ravi Sharma, S.V.S. Raju*

Department of Entomology & Agriculture Zoology, Institute of Agricultural Science, Banaras Hindu University, Varanasi- 221 005

Received – April 22, 2019; Revision – May 27, 2019; Accepted – June 07, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).289.294

KEYWORDS ABSTRACT

Chilli Insect pests continue to pose a major threat for achieving higher production of chilli crop. The experiment was conducted to study population dynamics of chilli thrips and aphids at Varanasi on 100 Environmental effects m2 unsprayed field during 2016-17. The chilli thrips and aphid population were counted from ten Population fluctuation randomly selected chilli plants and the abiotic factors like temperature, relative Humidity (RH), rainfall were also recorded. Result of the study revealed that chilli thrips population first appeared during the Chilli thrips third week of September and reached its peak during the third week of October. However, the incidence of the aphid population commenced from the fourth week of September and attained peak population Aphid levels during the fourth week of October. Further the correlation analysis of abiotic factors with chilli thrips population showed that the positive correlation with maximum temperature whereas, a negative correlation with relative humidity during morning, relative humidity during evening, minimum temperature and rainfall were also observed. While the aphid population showed positive correlation with maximum temperature. However, negative correlations were observed with relative humidity during morning, relative humidity during evening, rainfall and minimum temperature. These results of present study help in the development of the forecasting model and timely preparedness to manage pest problems and prevent crop losses.

* Corresponding author All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (S.V.S. Raju) Creative Commons Attribution-NonCommercial 4.0

International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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Influence of environmental factors on the population dynamics of chilli thrips, Scirtothrips dorsalis (Hood) and aphid, Aphis gossypii (Glover) 290

1 Introduction in the development of the forecasting model which is a useful tool to predict likely incidence of the insect pests on the crop. In this Chilli (Capsicum annuum L.) which is an important vegetable regard, the present investigation was carried out to estimate cash crop in India and is grown for the demand of its pungent population dynamics of chilli thrips and aphids on chilli in fruits, both green and ripe (latter dried form) to added pungency to relation to weather parameters. the food. As a condiment, it has become indispensable in every Indian home. It is also used medicinally, and in chutneys and 2 Materials and Methods pickles (Das, 2013). The world consumption of chillies and paprika is going up due to the increasing popularity of ethnic 2.1. Experimental site foods. India is the largest producer as well as consumer of chilli in The experiment was conducted during the year 2016-17 at the the world with the production of 3634 (in ̍000 MT) but its Vegetable Research Farm, B.H.U., Varanasi, India. Varanasi lies production pattern is highly erratic (Anonymous, 2017). Although between 24° 56’ N to 25° 35’ N Latitude and 82° 14’ E to 83° 24’ the crop has excellent export potential in addition to national E Longitude and the elevation is 82 m above the mean sea level, demand, it contributes to its low productivity through multiple almost in the centre of Indo-Gangetic belt and it possesses sub- limiting factors. The sustainability in chilli production is tropical climate. Pure seeds of the test variety, Bhagyalakshmi (G- threatened by many biotic stresses such as several insect pests and 4) were raised in the nursery on the experimental site on 3rd diseases (Hanumanthappa et al., 2018). Insect pests continue to August 2016. Other care was taken as per the recommendations to pose a major threat for achieving higher production of chilli crops maintain the nursery under unsprayed conditions. and about 57 species of pests were recorded by Reddy & Puttaswamy (1984) which attacked on chilli crop and causes yield 2.2. Field preparation and transplanting losses to range between 50 to 90 per cent (Rajput et al., 2017). Summer ploughing was given to the main field with the aim of The results of the survey conducted by Asian Vegetable Research exposing eggs and another stage of harmful insect pests and and Development Centre (AVRDC) indicated that the major under-ground reproductive parts of weeds. A bulk plot of 100 m2 sucking insect pests that attack chilli are thrips (Scirtothrips was raised to study the population dynamics of chilli thrips and dorsalis Hood) and aphids (Myzus persicae Sulzer, Aphis gossypii aphid. Thirty days old seedlings were transplanted in the main Glover) in Asia (Anonymous, 1987). Chilli thrips, S. Dorsalis is field on 2nd September 2016 with a plant spacing of 60×45 cm. All regarded to be one of the most damaging pests and may lose 30 to the recommended agronomic practices like weed control, fertilizer 50% of the plant under serious infestation (Bhede et al., 2008). application and irrigations were followed except for chemical Thrips can cause damage to chilli directly by feeding on leaves, treatment of the experimental plot. fruits or flowers. Feeding injury from thrips on leaves may affect leaf size, affect carbon allocation in the plant (Welter et al., 1990; 2.3. Pest sampling Shipp et al., 1998) and reduce photosynthetic capacity (Tommasini & Maini, 1995). Thrips also cause indirect damage The observations of pest population were recorded in these by transmitting the number of plant virus genera (Jones, 2005). unprotected plot at 7 days interval (Standard weeks) from 7 days The aphid is also one of the most important constraints in chilli after transplanting (DAT) up to crop harvesting. The population of production. Aphid became the most destructive insect pests of chilli thrips and aphids were counted on fully opened leaves from chilli in India and did huge economic damage. One recent study ten randomly selected plants and three leaves (One from the top, reported that severely infested chilli plant almost failed to produce middle and lower canopy) from each plant were taken for any fruits or produced a bare minimum number of deformed fruits recording the pest population. The population of both nymphs and (Shi et al., 2011). Under favorable conditions, aphid reproduces at adults were counted early in the morning before 8 AM when the a faster rate and may cause damage to 15-30% of total chilli pests were not much active. production (Das, 2013). Climatic factors such as temperature, relative humidity and rainfall are also known to have a significant 2.4. Meteorological data influence on insect population fluctuation (Sharma et al., 2018). The basic information on the relative occurrence and population Weather data were recorded simultaneously from the metrological dynamics are necessary before deciding the strategy for observatory available at agricultural research farm, institute of management of any insect pest (Manjunatha et al., 2001; Meena et agricultural Sciences, BHU, Varanasi and correlated with the al., 2013). Knowledge of the seasonal abundance and population occurrence of the pest population. A simple correlation coefficient build up trend is essential to ensure timely preparedness to method was adopted to work out the relationship between the manage pest problems and prevent crop losses and it also to help occurrence of the pest and the weather parameters.

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291 Raju et al.

2.5. Statistical analysis i.e., at 12 days after transplantation with a mean population of

0.28 thrips per leaf and the corresponding average maximum and Weekly data of pest population was correlated with the prevailing minimum temperature prevailed during the initial infestation were climatic factors such as maximum temperature, minimum 31.20C and 25.60C, respectively and average morning and evening temperature, morning and evening relative humidity and rainfall relative humidity was 92% and 82%, respectively. The population was analyzed by calculating respective “r” (correlation of thrips increased from 39th SW and reached a peak level (4.47 coefficient) with the help of SPSS (Statistical Package for Social thrips/leaf) on 43th SW (Figure 1). The pest population had a

Sciences) software. gradual decline after 43rd SW and reached its minimum population (0.21 thrips / leaf) during 1st SW, corresponding to a peak and 3 Results minimum temperature of 20.10 0C and 11.60 0C respectively, 3.1. Chilli thrips while the relative moisture of morning and evening was 95% and 76% respectively. Table 2 showed that the correlation between The data present in Table 1 revealed that the chilli thrips chilli thrips population and the major weather parameters. The population was initially observed on 38th Standard Week (SW) findings disclosed that the population of chilli thrips showed a

Table 1 Seasonal incidence of chilli thrips and aphids on chilli during 2016-17

Standard Rainfall Temperature 0C Relative humidity % Mean no. of Mean no. of Month- date week mm Max. Min. Morn. Even. thrips/ leaf aphid/leaf 36 Sep 02-08 31.1 31.6 26.2 90 80 0 0 37 09-15 2.6 31.4 26.4 91 80 0 0 38 16-22 64.8 31.2 25.6 92 82 0.28 0 39 23-29 115.8 28.6 24.0 95 88 0.21 0.38 40 30-06 2.6 32.4 26.3 88 74 1.09 0.54 41 Oct 07-13 1.5 32.0 23.4 87 61 2.28 2.48 42 14-20 0.0 32.4 18.4 74 43 3.17 3.25 43 21-27 0.0 32.4 17.9 74 43 4.47 4.65 44 28-03 0.0 31.4 16.6 77 43 3.89 6.87 45 Nov 04-10 0.0 29.2 15.3 80 45 3.06 5.14 46 11-17 0.0 29.0 13.8 77 42 3.25 4.81 47 18-24 0.0 27.3 11.7 72 42 2.87 4.32 48 25-01 0.0 25.4 13.2 79 56 2.54 3.56 49 Dec 02-08 0.0 20.3 16.3 94 78 0.95 2.45 50 09-15 0.0 20.2 10.0 94 73 0.48 2.01 51 16-22 0.0 23.3 9.8 89 50 0.54 1.67 52 23-31 0.0 20.5 10.9 94 69 0.34 0.84 1 Jan 1-7 0.0 20.1 11.6 95 76 0.21 0.69 2 8-14 0.0 20.7 8.2 91 44 0.00 0.48 3 15-21 0.0 23.0 8.8 90 49 0.00 0.31 Max.- Maximum, Min.- Minimum, Morn.- Morning, Even.- Evening

Table 2 Correlation Coefficient (r) of chilli thrips and aphids on chilli with prevailing weather parameters during 2016-17

Maximum Morning relative Evening relative Parameters Minimum temperature Rainfall Temperature humidity humidity Chilli thrips 0.484* -0.042 -0.912** -0.693** -0.326

Aphids 0.255 -0.243 -0.817** -0.703** -0.377 *Correlation is significant at P≤0.05; **Correlation is significant at P≤0.01

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Influence of environmental factors on the population dynamics of chilli thrips, Scirtothrips dorsalis (Hood) and aphid, Aphis gossypii (Glover) 292

140 8 Mean no. of thrips/leaf Mean no. of aphid/leaf Rainfall

Max. Temp Min. Temp. Morn. RH 120 7

6 100

5 80

4 60 Abiotic factors Abiotic 3

40 2 population insects of no. Mean

20 1

0 0 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 1 2 3

Standard meteorological weeks

Figure 1 Influence of abiotic factors on the population chilli thrips and aphid positive significant correlation with maximum temperature The correlation between population of chilli thrips and abiotic (r = 0.484). Again, there was a negative significant correlation of factors shown in Table 2. The results indicated that the correlation pest count with morning relative humidity (r = -0.912) and between the aphid population and maximum temperature evening relative humidity (r = -0.693) and a negative non- (r = 0.255) was positive but non-significant while, negative non- significant correlation with minimum temperature (r = -0.042) and significant correlation was observed with minimum temperature rainfall (r = -0.326). (-0.243). Further, a negative significant correlation was reported between the pest population and morning relative humidity 3.2. Aphids (r = -0.817) and evening relative humidity (r = -0.703) and a negative non-significant correlation with rainfall (-0.377) were The data presented in Table 1 revealed that the incidence of aphid also notice during the experimental period. was initially observed on 39th SW i.e., at 18th day after transplantation with a mean population of 0.38 aphids per leaf and 4 Discussion and Conclusion corresponding average maximum and minimum temperature prevailed during the initial infestation were 28.60C and 24.00C, The results of the present investigation showed that the incidence respectively and morning relative humidity and evening relative of thrips population was commenced during the 3rd week of humidity was 95% and 88%, respectively. The population of September and the pest population reached its peak level during aphid had increased from 39th SW with 0.38 aphids/leaf and the 4th week of October. Earlier, Patel et al. (2009) and Barot et al. attainted its peak level (6.87 aphids/leaf) on 44th SW (Figure 1). (2012) reported similar observation that the incidence of S. After 44th SW, the pest population gradually declined and reached dorsalis on chilli crop commenced from the first week of its minimum of 0.31 aphids/leaf during 3rd SW and the September and continue up to the harvest of the crop being peak corresponding peak and minimum temperatures were 23.0 0C and activity was recorded in November. Further, Meena et al. (2013) 8.8 0C respectively, while the relative humidity in the morning reported that peak population of the thrips occurred during the 41th and evening was 90% and 49% respectively standard week (October) and Chandra & Rana (2014) also

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293 Raju et al. observed that the population of S. dorsalis on chilli crop attained its temperature showed a positive impact on the population of chilli peak at the beginning of autumn season (first half of October 2013). thrips and aphid, while relative humidity and rainfall showed a The correlation between population of chilli thrips and abiotic negative impact on the population of chilli thrips and aphid. These factors shown in Table 2. The chilli thrips populations during the findings could help in the development of the forecasting model season revealed a significant positive correlation with maximum which will be a useful tool to predict likely incidence and temperature while, non- significant negative correlation with intensity of the insect pests on the crop to ensure timely minimum temperature and rainfall. However, a significant preparedness to manage pest problems and prevent crop losses. negative correlation observed with morning and evening relative humidity. The results are in close accordance with Pathipati et al. Acknowledgement (2014) who also observed that the thrips population had a negative correlation with minimum temperature, morning and evening The authors are thankful to Government of Uttar Pradesh for relative humidity, rainfall and positive correlation with maximum providing scholarship and Institute of Agricultural Sciences, temperature. Further, Asma & Hanumantharaya (2015) also BHU, Varanasi, for providing facilities to conduct the recorded a highly significant and positive correlation of thrips experiments. with maximum temperature. Lingeri et al. (1998) also reported that thrips population increased during the dry period with lower Conflict of Interest minimum temperature, while Kumar et al. (2006) and Meena et al. Authors would hereby like to declare that there is no conflict of (2013) reported that the thrips showed a negative correlation with interests that could possibly arise. relative humidity.

Whereas, the incidence of the aphid population was first observed References during the 4th week of September and reached to a peak during the Anonymous (1987) Progress Report for 1987, Asian Vegetable 44th standard week (October) thereafter pest population declined. Research and Development Centre, Pp. 77-79. These observations are in close accordance with the results obtained by Roopa & Kumar (2014) that the infestation of aphid Anonymous (2017) Agricultural statistics at a glance. Government started during the third week of September. The incidence was of India, Ministry of Agriculture, Department of Agriculture and observed throughout the cropping season and peak incidence was Cooperation, Directorate of Economics and Statistics. attained during the third and fourth week of October. These results are also in close confirmation with Bharadiya & Patel Asma A, Hanumantharaya L (2015) Population dynamics of (2005) who reported that the peak activity of aphid, A. gossypii , major insect and mite pests of chilli. Journal of Experimental during third week of November. Meena et al. (2013) observed that Zoology India 18: 173-176. the aphids appeared little late during the year in chilli crop. The correlation analysis of pest populations was positive non- Barot BV, Patel JJ, Shaikh AA (2012) Population dynamics of significant with maximum temperature. However, a negative and chilli thrips, Scirtothrips dorsalis Hood in relation to weather non- significant correlation was observed with minimum parameters. AGRES – An International e-Journal 1: 480-485. temperature and rainfall whereas, a negative and highly Bharadiya AM, Patel BR (2005) Succession of insect pests of significant correlation was found with morning relative humidity brinjal in north Gujarat. Pest Management and Economic Zoology and evening relative humidity. These results are in close 13: 159-161. association with the findings of Yadav et al. (2014) who reported a negative correlation between the temperature, humidity and Bhede BV, Suryawanshi DS, More DG (2008) Population rainfall with aphid population. Further, Debaraj & Singh (2004) dynamics and bio-efficacy of newer insecticide against chilli reported the negative correlation between temperature and aphid thrips, Scirtothrips dorsalis (Hood). Indian Journal of Entomology infestation. Roopa & Kumar (2014) also revealed that the aphid 70: 223-226. population exhibited a negative correlation with maximum temperature, minimum temperature and sunshine hours. Chandra M, Rana KS (2014) Seasonal Population Fluctuation of Thrips tabaci and Scirtothrips dorsalis (Thysanoptera: Thripidae) From the above findings it can be concluded that the peak period in Western Uttar Pradesh. Bulletin of Environment, Pharmacology of chilli thrips population was recorded in the third week of and Life Sciences 3: 135-140. October (43rd standard week) and the aphid population was recorded highest in the fourth week of October (44th standard Das G (2013) Efficacy of imidacloprid, a nicotinoid group of week). The correlation ananlysis with abiotic factors revealed that insecticide against the infestation of chilli aphid, Myzus persicae

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(Hemiptera: Aphididae). International Journal of Biology and Population Dynamics of Major Insect Pests Infesting Chilli Biological Sciences 2: 154-159. (Capsicum annum L.). International Journal of Pure Applied. Biosciences 5: 1465-1470. Debaraj Y, Singh TK (2004) Population dynamics of cabbage aphid Brevicoryne brassicae (Linn.) in relation to abiotic and Reddy DNR, Puttaswamy (1984a) Pest infesting chilli (Capsicum biotic factores at different altitudes of Manipur. Indian Journal of annuum L.) in the transplanted crop. Mysore Journal of Entomology 66: 172-175. Agriculture Science 19: 236- 237

Hanumanthappa PV, Chinnaswamy N , Annabathula MN, Reddy Roopa M, Kumar CTA (2014) Seasonal incidence of pests of RS, Ramachandrappa NN (2018) Morphometric and molecular capsicum in Bangalore conditions of Karnataka, India. Global diversity among the isolates of colletotrichum species causing Journal of Biology, Agriculture and Health Sciences 3: 203-207. anthracnose disease of chilli. Journal of Experimental Biology and Agricultural Sciences 6: 124 – 130 Sharma KR, Raju SVS, Roshan DR, Jaiswal DK (2018) Effect of abiotic factors on yellow stem borer, Scirpophaga incertulas Jones DR (2005) Plant viruses transmitted by thrips. European (walker) and rice leaf folder, Cnaphalocrocis medinalis (guenee) Journal of Plant Pathology 113: 119–157. population. Journal of Experimental Zoology India 21: 233-236.

Kumar MR., Reddy KL, Lakshmi V, Reddy DR, Gour TB (2006) Shi X, Jiang L, Wang H, Qiao K, Wang D, Wang K (2011) Survey of thrips infesting roses and its relation with weather Toxicities and sublethal effects of seven neonicotinoid parameters. Indian Journal of Entomology 68: 197-202. insecticides on survival, growth and reproduction of imidacloprid- resistant cotton aphid, Aphis gossypii. Pest Management Sciences Lingeri MS, Awaknavar JS, Lingappa S, Kulkarni KA (1998) 67: 1528-1533. Seasonal occurrence of chilli mites (Polyphagotarsonemus latus (Banks) and thrips (Scirtothrips dorsalis Hood). Karnataka Shipp J, Hao X, Papadopoulos A, Binns M (1998) Impact of Journal Agriculture Science 11: 380-385. western flower thrips (Thysanoptera: Thripidae) on growth, photosynthesis and productivity of green house sweet pepper. Manjunatha M, Hanchinal SG, Reddy GVP (2001) Survey of Scientia Horticultura 78:87–102 yellow mite and thrips on chilli in North Karnataka. Insect Environment 6: 178. Tommasini M, Maini S (1995) Frankliniella occidentalis and other thrips harmful to vegetable and ornamental crops in Europe. Meena RS, Ameta OP, Meena BL (2013) Population dynamics of In: Loomans AJM, van Lenteren JC, Tommasini MG, Maini S, sucking pests and their correlation with weather parameters in Ruidavets J (eds) Biological control of thrips pests, vol. 95, chilli, Capsicum annum L. crop. The Bioscan 8: 177-180. Wageningen University Papers, Wageningen, Pp. 1–42 Patel BH, Koshiya DJ, Korat DM (2009) Population dynamics of chillithrips, Scirtothripsdorsalis Hood in relation to weather Welter SC, Rosenheim JA, Johnson MW, Mau RFL, parameters. Karnataka Journal of Agricultural Sciences 22: 108-110. Gusukumaminuto LR (1990) Effects of thrips-palmi and western flower thrips (Thysanoptera, Thripidae) on the yield,growth, and Pathipati VL, Vijayalakshmi T, Naidu LN (2014) Seasonal carbon allocation pattern in cucumbers. Journal of Economic incidence of major insect pests of chilli in relation to weather Entomology 83:2092–2101 parameters in Andhra Pradesh. Pest Management in Horticultural Ecosystems 20: 36-40. Yadav AK, Acharya VS, Kashyap P, Meena VS, Singh SP (2014) Population dynamics of major sucking pests on chilli and its Rajput VS, Prajapati B.G, Pareek A, Patel PS (2017) Studies on relationship with weather factors. Annals of Horticulture 7: 103-108.

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Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 295 – 300

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

EFFECTIVE MANAGEMENT STRATEGY OF LITTLE LEAF DISEASE IN BRINJAL (Solanum melongena L.)

Rakesh Pandey1, A.K. Chaturvedi2

1Department of Entomology, Banda University of Agriculture & Technology, Banda (India) 2Krishi Vigyan Kendra, Bhadohi (ICAR-Indian Institute of Vegetable Research, Varanasi) (India)

Received – April 18, 2019; Revision – May 21, 2019; Accepted – June 08, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).295.300

KEYWORDS ABSTRACT

Brinjal Little leaf disease is distributed throughout the brinjal growing tracts. Farm experiments were conducted at five different locations with the technology developed by ICAR-Indian Institute of Vegetable Little leaf disease Research, Varanasi during two consecutive years i.e., 2014-15 and 2015-16. The technologies assessed Phytoplasma were FP (Farmers‟ practice): no use of chemicals to manage little leaf disease, only use chemicals viz., Profenophos, Cypermethrin etc. for the management of shoot and fruit borer; SDS: seedling treatment Integrated Pest Management for 20-30 minutes with (Streptomycin Sulphate + Tetracyclin hydrochloride @ 150 ppm) + destruction of the infected plants + need based foliar application of Streptomycin Sulphate + Tetracyclin Farmers‟ practice hydrochloride @ 150 ppm and Imidaclopirid @ 0.3 ml/l and STDS: SDS + installation of yellow sticky traps @ 15/ha after 20 days of transplanting for the management of vector i.e., leafhoppers. There were Varanasi 46.27 to 57.63 per cent plants infested in FP (farmers‟ practice). However, 33.55 to 43.88 per cent and 18.95 to 28.58 per cent plants were infested in SDS and STDS, respectively. The highest yield was observed in STDS (39.51 to 48.10 t/ha) followed by SDS (33.82 to 39.94 t/ha) and FP (23.26 to 32.88 t/ha) with the highest benefit cost ratio of 4.99 to 5.86 in STDS followed by 4.51 to 5.45 in SDS and 3.86 to 3.90 in FP (farmers‟ practice). It is evident from the study that STDS not only increased the yield from 46.29 to 69.83 per cent but also increased net return from 57.86 to 89.35 per cent in comparison to farmers‟ practice.

* Corresponding author All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Rakesh Pandey) Creative Commons Attribution-NonCommercial 4.0

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Effective management strategy of little leaf disease in brinjal 296

1 Introduction increases the chances of insecticide residues in the fruit.

Considering the principles of Integrated Pest Management (IPM), Brinjal, Solanum melongena L. is one of the most widely grown the present study was undertaken with the objective to investigate vegetable crops of both tropics and subtropics of the world. It is the effective management strategy by seedling treatment, being grown extensively in India, Bangladesh, Pakistan, China, destruction of infected plants, application of yellow sticky traps Philippines, France, Italy and United States (Rathnamma & Patil, and need based foliar application of Streptomycin Sulphate + 2017). It is one of the most important solanaceous vegetable crops Tetracyclin hydrochloride and Imidaclopirid against little leaf in the Indian sub-continent (Srinivasan & Huang, 2008). It disease under field conditions. contributes about 8.12% of the total vegetable production and about 8.0% of the total area under vegetables in India (Indian 2 Materials and Methods Horticulture Database, 2011). It is highly productive and finds its place as the poor man‟s vegetable (Som & Maity, 2002). It is 2.1 Location of the study grown in almost all parts of India, except in higher altitudes The study was conducted in 0.125 ha area located in Bhadohi throughout the year. Insect-pests and diseases are one of the (82056‟ east longitude and 25040‟ north latitude). The climate was important limiting factors in cultivation of brinjal. Little leaf hot and humid in summer and cold and dry in winter with an in disease is caused by phytoplasma and it causes significant losses between rainy season. The temperature in the area ranged between (Mitra, 1993). Stunted plant growth, shortening of internodes, 50C to 460C and an annual rainfall of 1563 mm was reported reduced leaf size and proliferation of shoots are the prominent (Singh et al., 2008). symptoms of infected plants. Several biological aspects of brinjal little leaf disease was reported by Varma et al. (1969) and Mitra 2.2 Details of technologies assessed against little leaf disease (1993). Plants infected with phytoplasmas exhibit symptoms suggesting a profound disturbance in the normal balance of The different insecticides are the only control measures adopted growth regulators, leading to virescence/ phyllody i.e., by the farmers (FP). To find out a safe, effective, economical and development of green leaf like structures instead of flowers, sustainable strategy for the management of little leaf disease, two sterility of flowers, proliferation of axillary buds resulting in technologies were formulated on the basis of researches “witches broom” symptoms, abnormal internode elongation and conducted at ICAR-Indian Institute of Vegetable Research, generalized stunting (Bertaccini, 2007). The insects of families Varanasi. Cicadellidae, Cixidae, Psyllidae, Delphacidae and Derbidae are responsible for transmission of phytoplasma (Weintraub & FP : Farmers‟ practice (no use of chemicals to Beanland, 2006). Bindra & Singh (1969) reported that the manage this disease, only use chemicals viz., Hishimonus phycitis Distant is the most common vector of this Profenophos, Cypermethrin etc. for the disease. management of shoot and fruit borer)

The field surveys indicated that the incidence of little leaf disease SDS : Seedling treatment for 20-30 minutes with was more than 50 per cent in the study area, however, Rathnamma (Streptomycin Sulphate + Tetracyclin & Patil, 2017 also reported up to 95 per cent incidence in brinjal hydrochloride @ 150 ppm) + destruction of the growing area. Farmers usually spray insecticides viz., infected plants + need based foliar application Profenophos, Cypermethrin, etc. once or twice at weekly intervals of Streptomycin Sulphate + Tetracyclin only to control the brinjal shoot and fruit borer (Pandey et al., hydrochloride @ 150 ppm and Imidaclopirid @ 2016). There were no concerted remedies against this serious 0.3 ml/l malady used by the farmers of the region. However, in India STDS : SDS + installation of yellow sticky traps @ According to Alam et al. (2006), more than 95% of the farmers 15/ha after 20 days of transplanting for the applied more than 40 sprays per season (in Gujrat) and 86% management of vector (trap was replaced at sprayed their crops twice or three times a week (in Uttar Pradesh) monthly interval) against eggplant shoot and fruit borer. Sole reliance on chemicals for the control of L. orbonalis made eggplant cultivation 2.3 Field trials uneconomical and also caused residual toxicity (Chandra et al., 2014). Moreover, despite serious damaging nature of little leaf Seedlings of eggplant were planted on raised beds in the first disease, its control tactic by and large is limited to frequent sprays week of July, 2014 & 2015. The improved variety New Kiran was of chemical insecticides in this region. Such practices of grown in the field to test the effectiveness of different treatments. insecticides usage is detrimental to the environment, also The seed bed was lightly irrigated regularly for ensuring proper

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297 Pandey & Chaturvedi growth and the development of the seedlings. Thirty to thirty five The cost of production comprised of costs for seed, nursery days old seedlings were transplanted in the second week of raising, field preparation, transplanting, fertilizer application, August, 2014 & 2015 with a distance between row to row and irrigation, weeding and harvesting, costs for insecticides, plant to plant of 90×90 cm. All the agronomic practices were spraying, trap, installation of yellow sticky traps and uprooting of similar for three assessed technologies. The weeds were removed infested plants. The Indian rupees (INR) converted in to US dollar mechanically twice at 30 and 60 days after transplanting with a (USD) by conversion rate i.e., 1 USD = 67.75 INR. small spade. The infested plants were uprooted by hand when symptoms of the disease appeared in field. The application of To compute the total return, the value of fruits obtained in each insecticides was done by back pack sprayers. treatment was calculated separately as per the market rate. The market price of eggplant was at USD $ 103.32/t (2014-15) and at Field trials were conducted in two successive cropping seasons USD $ 147.60/t (2015-16). Net return was also calculated by (2014-15 and 2015-16) at farmers‟ fields. The field trials were subtracting the total cost from total return. carried out in a randomized complete block design with five 2.5 Statistical analysis replications. The unit plot size was 12×7 m for each regime where the seedlings were transplanted. The data collected from the experiments were subjected to analysis of variance for different treatments. Fisher's protected 2.4 Data collection critical difference (CD) test was used to indicate the difference The date of pheromone traps installation (in September, 2014 & between the treatments at the probability level of p < 0.05 2015 during vegetative stage), the observations of trap catches following the procedure described by Gomez & Gomez (1984). were recorded at weekly intervals throughout the growing season 3 Results (ended the last week of March, 2015 & 2016 - full maturity of the crop). Ten plants were randomly selected from each plot and The effect of different disease management strategies on the tagged for the periodical observations on yield. Starting with the disease infested plants was significant during both the years first picking/harvesting (in the second week of October, 2014 & (Table 1). Maximum infested plants were recorded in FP 2015 - initiation of fruiting), fruits were weighed separately from (57.63%) followed by SDS (43.88%) and STDS (28.58%), each plot at each harvest (ended last week of March, 2015 & 2016 respectively, during 2014-15. However, there was 23.86% disease - full maturity of the crop). The infested plants were marked on infested plants prevented over farmers‟ practice in SDS and the basis of typical symptoms of the disease. The percent plant 50.40% in STDS during 2014-15. Similarly, little leaf disease infestation was assessed on the basis of recording the number of infested plants were maximum (47.27%) in FP and minimum plants showing disease symptoms and the total number of plants (18.95%) in STDS with a 29.02% in SDS and 59.91% prevention examined at each location throughout the cropping period by in STDS during 2015-16. using the following formula:

Number of infested plants A significant effect of different treatments on brinjal yield was Percent plant infested = × 100 Total number of plants reported doe both years. In 2014-15, maximum yield of 48.10 t/ha was recorded in STDS followed by SDS (39.94 t/ha) and FP The percent plant infestation was recorded separately per plot. (32.88 t/ha) with an additional gain over FP, 21.47% in SDS and The plot yield of each harvesting was also recorded separately and 46.29% in STDS (Table 2). However, STDS was significantly total yield was calculated in tonnes per hectare. The economic superior to SDS and FP. Similar observations were recorded parameters i.e., cost of cultivation, gross return, net return and during 2015-16 and maximum yield was recorded in STDS (39.51 benefit cost ratio were also assessed. To justify the economic t/ha) followed by SDS (33.82 t/ha) and FP (23.26 t/ha). An viability of the appropriate management strategy against little leaf additional gain of 45.40% in SDS and 69.86% in STDS were also disease, the benefit: cost (B: C) ratio was calculated from the recorded over farmers‟ practice. The lowest yield was recorded marketable yield, regarding cost of treatments incurred in the from FP during both years (Table 2). management. The market price of eggplant fruits, rate of insecticides and labor cost were undertaken as approved by the Overall economic assessment for the tested management Government to compute the B: C ratio by using following formula strategies showed that both years followed similar trend (Table 3). (Baral et al., 2006): The highest total return in 2014-15 was USD $ 4969.90/ha (STDS), followed by USD $ 4126.91/ha (SDS) and USD $ Value of yield over control (USD $/t) BC ratio = 3397.26/ha (FP); and in 2015-16 USD $ 5831.97/ha, USD $ Total cost of production (USD $/ha) 4992.57/ha and USD $ 3433.91/ha for STDS, SDS and FP,

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Effective management strategy of little leaf disease in brinjal 298

Table 1 Percent plant infestation due to little leaf disease and percent control over farmers‟ practice

2014-15 2015-16 Technologies assessed Plant infestation (%) Control over farmers‟ Plant infestation (%) Control over farmers‟ practice (%) practice (%) FP 57.63a - 47.27a -

SDS 43.88b 23.86 33.55b 29.02

STDS 28.58c 50.40 18.95c 59.91

LSD (CD) (P=0.05) 3.78 5.08

SE± 1.64 2.20

Table 2 Total marketable yield of brinjal and percent gain over farmers‟ practice

2014-15 2015-16 Technologies assessed Yield (t/ha) Gain over farmers‟ practice (%) Yield (t/ha) Gain over farmers‟ practice (%) FP 32.88c - 23.26c -

SDS 39.94b 21.47 33.82b 45.40

STDS 48.10a 46.29 39.51a 69.86

LSD (CD) (P=0.05) 1.93 2.17

SE± 0.84 0.94

Table 3 Overall economic assessment of field trials

Year Benefit : Cost assessments FP SDS STDS

2014-15 Cost of production (USD $) 879.40 915.27 994.97

Total Return (USD $)1 3397.26 4126.91 4969.90

Net Return (USD $) 2517.86 3211.64 3974.93

Benefit / Cost ratio 3.86 4.51 4.99

2015-16 Cost of production (USD $) 879.40 915.27 994.97

Total Return (USD $)2 3433.91 4992.57 5831.97

Net Return (USD $) 2554.51 4077.30 4837.00

Benefit / Cost ratio 3.90 5.45 5.86

1 Market price of eggplant at USD $ 103.32/t during 2014-15. 2 Market price of eggplant at USD $ 147.60/t during 2015-16. respectively. The benefit: cost ratio was observed to be higher in plants is growing rapidly in brinjal growing tracts. Azadvar & STDS (4.99) followed by SDS (4.51) and FP (3.86) during 2014- Baranwal (2012) observed only 2-4 per cent in New Delhi. Little 15 and STDS (5.86), SDS (5.45) and FP (3.90) during 2015-16. leaf disease symptoms were observed in approximately 20 per The highest additional income over FP (the difference of net cent of the brinjal (S. melongena) in a survey in Bihar (Kumar et return between STDS and FP) was USD $ 1457.06/ha and USD $ al., 2012). The diseased plants exhibited proliferation of branches 2208.69/ha during 2014-15 and 2015-16, respectively. with shortened internodes and reduced-size leaves which gave rise to the little leaf appearance (Raj et al., 2008). It revealed that there 4 Discussion & Conclusion was 47.27 to 57.63 percent plants were infested with little leaf disease. However, Rathnamma & Patil (2017) reported up to 95 The severity of the little leaf disease, shortening of internodes, per cent incidence of the little leaf disease in brinjal growing areas excessive branching giving witches‟ broom appearance on brinjal of Karnataka. Different studies over the years showed that there is

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299 Pandey & Chaturvedi an increasing trend of spreading little leaf disease in brinjal There is scanty literature pertaining to the study, however, the growing tracts. available literature is in the line of confirmation of the findings. Sole reliance on chemical insecticides was found to be highly Due to catch the better market price, the farmers transplant the uneconomical (Baral et al., 2006; Srinivasan, 2008) as less return brinjal early that is also one of the important pre-disposing factors was obtained against per rupee invested. This was mainly due to of this disease. Therefore, different disease management strategies the high cost of insecticide and the labor charges engaged in their were evaluated so that farmers fetch a good income from sprayings. It is evident from the study that the seedling treatment cultivation of brinjal. ICAR-Indian Institute of Vegetable for 20-30 minutes with (Streptomycin Sulphate + Tetracyclin Research, Varanasi has recommended the use of tetracycline @ hydrochloride @ 150 ppm) + installation of yellow sticky traps @ 100 ppm (Pandey et al., 2003) to manage the little leaf disease. 15/ha after 20 days of transplanting + destruction of the infected Bindra et al. (1972) also reported that plants treated with 500 ppm plants + need based foliar application of Streptomycin Sulphate + of the tetracycline resumed normal growth with respect of leaf Tetracyclin hydrochloride @ 150 ppm and Imidaclopirid @ 0.3 size and length of internodes 30 to 37 days after the last spray. In ml/l for the management of vector not only increased the yield other treatments i.e., 100, 250 ppm recovery was also observed from 46.29 to 69.83 per cent but also increased net return from but it was not pronounced. However, disease symptoms started 57.86 to 89.35 per cent in comparison to farmers‟ practice. reappearing 16 to 63 days after the last spray i.e. 14 sprays of the tetracycline suppressed the symptoms only for 20 to 30 days. The Acknowledgements disappearance of symptoms after antibiotic (i.e., tetracycline) treatment provided additional evidence to support the diagnosis The authors‟ gratefully acknowledge the financial support and (Lee & Davis, 1992). A minimum incubation period of 15 days encouragement from Directors, ICAR-Indian Institute of was observed to be essential in the vector for successful Vegetable Research, Varanasi and ICAR-Zonal Project transmission and optimum incubation period of the mycoplasma Directorate Zone – IV, Kanpur. in the host plant was recorded to be 35 days. In nature, the brinjal Conflict of Interest plants are likely to be infected in the nursery or after transplanting, a properly devised method for infiltrating the plants Authors would hereby like to declare that there is no conflict of with tetracyclines before transplanting would eliminate the interests that could possibly arise. chances of carrying the little leaf disease from nursery to field (Varma et al., 1969). References

Brinjal little leaf disease is transmitted by insects (leaf hopper) such Alam SN, Hossain MI, Rouf FMA, Jhala RC, Patel MG, Nath LK, as Hisimonus phycitis which is common on brinjal crop (Singh et Sengupta A, Baral K, Shylesha AN, Satpathy S, al., 2014; EFSA Panel on Plant Health, 2017). Further, Srinivasan & Shivalingaswamy TM, Cork A, Talekar NS (2006) Control of Chelliah (1977) reported that transmission of little leaf mycoplasma eggplant fruit and shoot borer in South Asia. Technical Bulletin of S. melongena by H. phycitis revealed that the minimum 36, AVRDC – The World Vegetable Center, Shanhua, Taiwan. acquisition and inoculation feeding period was one hour each while the optimum was 24 and 48 hours, respectively. For the Azadvar M, Baranwal VK (2012) Multilocus sequence analysis of management of leafhopper, imidacloprid has been used. Mhaske & phytoplasma associated with brinjal little leaf disease and its Mote (2005) found that imidacloprid (18 g/ ha), thiamethoxam (25 detection in Hishimonus phycitis in India. Phytopathogenic g/ha) were effective against jassids (Amrasca bigutulla bigutulla) up Mollicutes 2:15-21. to 14 days after spray. Foliar application of imidacloprid caused Baral K, Roy BC, Rahim KMB, Chatterjee H, Mondal P, Mondal immediate and long-lasting reductions in control potato leafhopper, D, Ghosh D, Talekar NS (2006) Socio-economic parameters of Empoasca fabae (Harris) (van Timmeren et al., 2011). Yellow pesticide use and assessment of impact of an IPM strategy for the sticky trap is also an added advantage to manage the leaf hoppers. control of eggplant fruit and shoot borer in West Bengal, India. - When the crop is in vegetative stage there is normally no sign of Technical Bulletin No. 37. AVRDC publication number 06-673. incidence of little leaf disease observed. However, in reproductive AVRDC-The World Vegetable Center, Shanhua, Taiwan, Pp. 36. phase when the canopy is in full swing the multiplication of leaf hoppers takes place rapidly consequently, the incidence of the Bertaccini A (2007) Phytoplasmas: diversity, taxonomy, and disease also aggravates. During multiplication of leaf hoppers, the epidemiology. Frontiers in Bioscience 12: 673–689. yellow sticky trap acts as barrier to move away between plants and thus reducing the population of leaf hoppers and minimizing the Bindra OS, Singh B (1969) Biology and bionomics of Hishimonus little leaf disease incidence. phycitis (Distant), a jassid vector of little leaf disease of brinjal

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(Solanum melongena L.). Indian Journal of Agricultural Sciences orbonalis (Lepidoptera: Crambidae). Journal of Crop Protection 5: 39: 912-919. 553-563. doi: 10.18869/modares.jcp.5.4.553

Bindra OS, Sohi SA, Khatri HL, Deol SG (1972) Effect of Raj SK, Snehi SK, Kumar S, Khan MS (2008) First finding of achromycin (Tetracycline hydrochloride) on brinjal little leaf 'Candidatus Phytoplasma trifolii' (16SrVI group) associated with little pathogen. Current Science 41: 819-820. leaf disease of Datura inoxia in India. New Disease Reports 18: 44.

Chandra S, Mahindrakar AN, Fugare MK, Shinde LP (2014) Rathnamma, Patil MS (2017) Survey for Little Leaf of Brinjal Studies on persistence pattern of pesticides on brinjal. Disease (Candidatus Phytoplasmatrifolii) Incidence in Northern International Journal of Current Research in Chemistry and Karnataka. International Journal of Current Microbiology & Pharmaceutical Sciences 1: 88-91. Applied Sciences 6(11): 1248-1253. doi: https://doi.org/10.20546/ijcmas.2017.611.148 Gomez KA, Gomez AA (1984) Statistical Procedures for Agricultural Research, second ed. John Wiley and Sons, New Singh AK, Singh L, Singh A, Singh RK (2008) District specific York, NY, USA. technological interventions for eastern region. In: Inventory of Agricultural Technologies for Uttar Pradesh, Zonal Coordination Indian Horticulture Database (2011) National Horticulture Board, Unit, Zone IV, Indian Council of Agricultural Research, Pp. 93. Ministry of Agriculture, Govt. of India, Pp. 278. Singh BK, Singh S, Singh BK, Yadav SM (2014) Some Important EFSA Panel on Plant Health (PLH), Jeger M, Bragard C, Plant Pathogenic Disease of Brinjal (Solanum melongena L.) and Caffier D, Candresse T, Chatzivassiliou E, Dehnen‐Schmutz K, their Management. Plant Pathology Journal, 13: 208- Gilioli G, Gregoire JC, Anton J, Miret J, Navarro MN, Niere B, 213.doi 10.3923/ppj.2014.208.213. Parnell S, Potting R, Rafoss T, Rossi V, Urek G, Bruggen AV, van der Werf W, West J, Winter S, Gardi C, Aukhojee Som MG, Maity TK (1986). Brinjal. In: Bose TK, Som MG M, Bergeretti F, MacLeod A (2017) Pest categorisation of (Eds.), Vegetable Crops in India, 3rd revised edition, Naya Hishimonus phycitis. EFSA Journal. 15. Prakash, Calcutta, pp. 293-335. 10.2903/j.efsa.2017.5037. Srinivasan K, Chelliah S (1977) Transmission studies on little leaf Kumar J, Gunapati S, Singh SP, Lalit A, Sharma NC, Tuli R mycoplasma of brinjal. Madras Agricultural Journal 64:94-98. (2012) First report of a „Candidatus Phytoplasma asteris‟(16Srl group) associated with little leaf disease of Solanum melongena Srinivasan R (2008). Integrated Pest Management for eggplant (brinjal) in India. New Disease Reports 26: 21. fruit and shoot borer (Leucinodes orbonalis) in south and south- east Asia: Past, Present and Future. Journal of Biopesticides 1: Lee IM, Davis RE (1992) Mycoplasmas which Infect Insects and 105-112. Plants. In: Maniloff J, McElmansey RN, Finch LR, Baseman JB (Eds), Mycoplasmas: Molecular Biology and Pathogenesis, Srinivasan R, Huang CC (2008) Effect of simulated borer infested American Society for Microbiology, Washington DC, pp. 609. shoot pruning on yield parameters of eggplants. Journal of Asia Pacific Entomology 12: 41-43. DOI: Mhaske BM, Mote UN (2005) Studies on evaluation of new https://doi.org/10.1016/j.aspen.2008.10.001. insecticides against brinjal pest complex. Journal of Maharashtra Agricultural Universities 30: 303-306. Van Timmeren S, John CW, VanderVoort C, Isaacs R (2011) Comparison of foliar and soil formulations of neonicotinoid Mitra DK (1993) Little leaf, a serious disease of eggplant insecticides for control of potato leaf hopper, Empoasca fabae (Solanum melongena). In: Management of plant diseases caused (Homoptera: Cicadellidae), in wine grapes. Pest Management by fastidious prokaryotes, edited by Raychaudhuri SP, Teakle DS Science 67: 56-567. Associated Publishing Co. New Delhi, India Pp. 73-78. Varma A, Raychaudhuri SP, Chenulu VV, Singh S, Ghosh SK, Pandey KK, Pandey PK, Chandra S (2003) Sabjiyo me ekikrit Prakash N (1969) Yellows Type of Diseases in India: Eggplant nashijeev prabandhan, ICAR-Indian Institute of Vegetable little leaf. Division of Mycology and Plant Pathology, Indian Research, Varanasi, Pp. 9-10. Agricultural Research Institute, New Delhi.

Pandey R, Chaturvedi AK, Chaudhary RP (2016) Effectiveness of Weintraub PG, Beanland L (2006) Insect vectors of phytoplasmas. IPM strategies against eggplant shoot and fruit borer Leucinodes Annual Review of Entomology 51: 91-111.

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Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 301 – 307

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

EFFICACY OF SILVER NANOPARTICLES SYNTHESIZED ON Commiphora gileadensis (BALSAM) EXTRACT AGAINST INFECTIOUS BACTERIA

Bassam Oudh Al johny

Department of Biological Science, Faculty of Science, King Abdulaziz University, PO Box 80203, Jeddah 21589, Kingdom of Saudi Arabia.

Received – April 10, 2019; Revision – May 21, 2019; Accepted – June 09, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).301.307

KEYWORDS ABSTRACT

Commiphora gileadensis Compare to the other metal ion, silver nanoparticles has wide application in the field of microbiology and pharmacology drug. The aim of present study was to synthesize cheap and eco-friendly silver Silver nanoparticles nanoparticles on Commiphora gileadensis extract and evaluate its antimicrobial properties against selected bacterial strains. Aqueous extract of C. gileadensis was used for the synthesis of silver E.coli nanoparticles. These synthesized nanoparticles were characterized by using UV spectrophotometer, Multi-drug resistant scanning electron microscope (SEM) and EDX. The characterization of AgNPs was carried out by uv- Staphylococcus aureus (MRSA) vis spectrum at λmax = 435 nm and the peak was correlated to the plasmonic nature of the particles as reported by number of earlier reports. In addition, EDX spectrum has been showed four peaks corresponding to the silver, zinc, carbon and oxygen. The results of present study indicated that the plant extract has very important role in the synthesis of AgNPs. Antibacterial activity of nanoparticles was evaluated using CFU method against E.coli O157:H7 and Methicillin-resistant Staphylococcus aureus (MRSA). Synthesized nanoparticles exhibit significant antibacterial activity against the infectious bacteria.

* Corresponding author All the articles published by Journal of Experimental Biology and Agricultural Sciences are licensed under a E-mail: [email protected] (Bassam Oudh Al johny) Creative Commons Attribution-NonCommercial 4.0

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Efficacy of silver nanoparticles synthesized on Commiphora gileadensis extract against infectious bacteria 302

1 Introduction 2016a; Kamal et al., 2017; Haider et al., 2018; Ali et al., 2018a;

Ali et al., 2018b). The AgNps have attained lots of attention Plant extracts are very important and many of them possess because of their physical and chemical properties i.e., excellent biological properties (Abdul-Ghani & Amin, 1997; conductivity, catalytic activity, stability, being non-reactive & Adebayo et al., 2014; Shaheen et al., 2017). Commiphora non-toxic and eco-friendly. Along with above mentioned gileadensis (syn. C. opobalsamum) is very common aromatic properties, AgNps have extremely exalted antifungal, anti- shrub and the Red sea area is known as its native land, which inflammatory, antiviral and antibacterial activities and can be used encompasses the territory of Saudi Arabia, Ethiopia, Oman, in field of medicine for treating and diagnosis many chronic and

Yemen and some other countries of this region (Wineman et al., acute diseases (Mittal et al., 2013; Bello et al., 2017b; Borros et 2015). C. gileadensis was first discovered by Carl Linnaeus from al., 2018; Singh et al., 2018). The AgNps have great application in Yemen and identified as “Balm of Gilead” (Amiel et al., 2012). It biomedical leading to the production of antiseptic sprays, is also known as balsam and find very useful for perfume industry dressing's wounds fabrics as well as antiseptic topical creams. (Miller et al., 1988). All parts of the balsam such as bark, wood, Antibacterial and antifungal properties of the AgNps have also seeds, extract (Al-sieni, 2013) and plant Sap (Iluz et al., 2010) are been reported, which demonstrated that AgNps disrupt cell frequently used in pharmaceutical industries. Oil containing membrane of the unicellular microbes leading to plasmolysis and balsam sap was considered of prime an incentive in getting ready hence kill the microbes (Khan et al., 2015; Wajid Ullah et al., medicinal balms (Iluz et al., 2010). According to Iluz et al. (2010), 2016). In addition, disruption of the cell membrane results sap of balsam has capacity to cure cerebral pains. Crude deterioration of membrane bound enzymes and cause deactivation methanolic extract of C. gileadensis showed significant of the enzymes, which cause death of microorganisms (Kamal et antibacterial activity against E. coli, and Bacillus cereus, with a al., 2016; Bello et al., 2017b). The efficacy of the AgNps is minimum inhibitory concentration of 62.5 μg/ml (Al-sieni., 2013). greatly dependent of pH, size, ionic strength and capping agent of nanoparticles (Ahmed et al., 2016b). The synthesis of AgNps Medicinal properties of this plant are well known and because of attained greater attention of the researchers because of their this it became active ingredient of many therapeutic drugs. For broader applications range and utilities in medicine and diagnosis instance, dried bark has been placed on wounds as a germicide, against variety of cancers (Kuppusamy et al., 2016). Present study while a tincture of ground balsam bark is utilized to treat various has been undertaken to assess the antimicrobial properties of skin sicknesses such as aggravations and dermatitis (Mittal et al., AgNps synthesized on C. gileadensis extract. Moreover, these 2013). Researcher also reported hypotensive impact of C. AgNps were synthesized against E.coli O157:H7 and a common gileadensis; oral administration of plant extract initiate muscarinic antibacterial drug-resistant Staphylococcus aureus (MRSA). cholinergic receptors of the mind, which initiate hypotensive impact which is at par the effect given by atropine sulfate (1-4 2 Materials and Methods mg/kg). In addition, the leaves of balsam plant were utilized by Arab populaces as pain relieving, purgative and diuretic operators 2.1 Culturing of Pathogenic Bacteria (Abdul-Ghani & Amin, 1997). Another investigation on rats revealed positive impact of plant extricate in the treatment of E.coli O157:H7 and multi drug-resistant Staphylococcus aureus tentatively delivered stomach ulcers. MRSA which used as model bacteria were provided by King Fahad Center, King Abdulaziz University, Jeddah, Saudi Arabia. As stated earlier that extract of C. gileadensis has been used These microorganisms were used for the evaluation of bactericidal effectively against the bacterial growth but its high dosage was activity of synthesized nanoparticles. Nutrient agar (Sigma- recommended. Therefore, it is utmost important to explore the Aldrich-70148) contains 5 g/L peptones, 2 g/L yeast extract, 1 g/L alternate ways of using this plant extract for the bacterial growth meat extract, 2 g/L sodium chloride and 15 g/L agar in deionized inhibition. One of the effective way of using plant extracts is to water was used for culturing these pathogenic bacteria. Media was utilize it for the synthesis of metal nanoparticles (Nps). In fact, prepared, autoclaved and pours into plates. Bacterial cells were metal Nps have been found highly significant in many studies transferred after solidification of media to culture plates. related to various scientific fields (Ahmed et al., 2016a; Ali et al., 2017a; Ali et al., 2017b). Nowadays, plant extracts based Nps 2.2 Antibacterial Assessment synthesis is one of the emerging field of nanotechnology. Various studies have reported the use of Silver nanoparticles (AgNps) in Colony forming unit (CFU) was used for the enumeration of medicine, consumer products, food-technology and in other bactericidal activity of silver nanoparticles synthesized using industrial products because of their exclusive chemical, catalytic, balsam plant extract. 100 µl of E.coli O157:H7 and multi-drug- sensing, wound healing and physical properties (Ahmed et al., resistant S. aureus (MRSA) were added to 10 ml of tube

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303 Bassam Oudh Al johny containing nutrient broth medium along with silver nanoparticles . The surface morphology and size of the AgNPs were analyzed by These tubes were incubated for 24 hours at 35 ̊C. The samples employing a Scanning electron microscope (SEM) and energy were serially diluted and then spreaded onto nutrient agar dispersive X-ray spectroscopy (EDX). The particle size distribution containing plates. Nutrient Agar plates were incubated for 24 and surface charge of AgNPs were determined using particle size hours in order to check colony formation. Colony counter was analyzer (Zetasizernano ZS) at 25 °C with 90° detection angle. used in order to count the bacterial colonies. Each treatment was repeated thrice. 3 Results and Discussion

2.3 Balsam extraction 3.1 Antibacterial study

A highly acclaimed method of obtaining the plant extract was used for Various properties of the AgNps such as cryogenic super this study. First, a whole plant of balsam was obtained from Baddar, conducting materials, composite fibers, food industry, cosmetic Madinah region, Saudi Arabia. Plant material cut into small pieces and products and in electronic gadgets have been well reported by transfer to mortar containing liquid nitrogen. These pieces were various researcher's. In present study, the plant extract assisted crushed into fine pieces using mortar and then incubate on magnetic AgNps have been used against the growth of two selected stirrer for 48 hours. Then filtered using filter paper concentrated and bacterial strains. Result of present study revealed that synthesized dried. This whole process was performed over a period of one week. AgNps have significant antibacterial activities and inhibiting the bacterial growth which is a major concern. Growth characteristics 2.4 Biosynthesis of AgNPs of E.coli O157:H7 and MRSA are shown in figure 1 & table 1.

Aqueous extract of balsam was treated with a solution of AgNO3. The treatment was performed in such a way that a solution of 1 Mm Control Silver nanoparticle of AgNO3 in final concentration was made. The mixture was stirred on a magnetic stirrer at 60 oC. Next, the synthesis AgNPs was indicated by color change (brown) of the solution. After two days, the solution was centrifuged and mixture was collected after discarding the supernatant. The collected AgNPs were washed with distilled water and air-dried for characterization and further studies. E.Coli E.Coli

2.5 Characterization of AgNPs Control Generally, the characterization of NPs required determining the size, shape, surface area and dispersity because its properties are dependent on these features. Formation of AgNPs was confirmed by Ultraviolet-visible spectral analysis (UV-vis) spectroscopy. The absorbance spectra were recorded using a UV-Vis spectrophotometer at a wavelength range of 300–700 nm. Fourier MRSA MRSA

Transform Infrared Spectroscopy (FTIR) was performed on FTIR Spectrometer to detect the possible functional groups in Figure 1 Growth of E. coliO157:H7 and Methiciallin-resistant Staphylococcus aureus (MRSA) on Nutrient, plate agar-showing biomolecules present in the plant extract. inhibition in the presence of AgNPs

Table 1 Effectiveness of the AgNps against bacterial strains

Incubation Periods Reduction (%) Treatments 12 Hrs (Cfu/ml) 24 Hrs (Cfu/ml) 12 Hrs (Cfu/ml) 24 Hrs (Cfu/ml)

Control (E.coli O157:H7) 76×106 102×106 0 0

E.coli O157:H7 + Ag Nps 0×106 0×106 100 100

MRSA (Control) 86×106 109×106 0 0

MRSA + Ag Nps 0×106 0×106 100 100

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Efficacy of silver nanoparticles synthesized on Commiphora gileadensis extract against infectious bacteria 304

Results of study revealed that control (without AgNps nanoparticles) did not have any inhibition activity after 24 hours, while silver nanoparticles showed highest inhibition in E.coli O157:H7 and MRSA multiplication. This enhanced activity may be attributed to the silver nanoparticles. As E.coli is gram-negative bacteria that has a thin peptidoglycan layer and has complex cell wall containing two cell membrane (Kamal et al., 2016). The cell membrane of this bacteria is negatively charged, which may interact with the positively charged silver nanoparticle. This interaction alters cell membrane properties by forming pits and blocking the intake of nutrients to the cell subsequently AgNPs penetrating inside bacterial cell, thereby causes

DNA damage (Khan et al., 2015). Other than this mechanism, these nanocomposite can also produce highly reactive species when heavy Figure 2 UV-vis spectrum of the synthesized Ag nanoparticles using metals react with proteins which leads to the inactivation of protiens, balsam plant extract (Experimental conditions: Conc. of AgNO3 (1mM), spectrum obtained after 48h) i.e. hydrogen peroxide, superoxide’s or hydrogen radicals. In addition, toxicity studies of AgNPs was observed against Gram-positive Staphylococcus aureus ATCC 35696 showed silver ions can interact with the thiol groups in proteins and inactivate them as well DNA molecules become condensed by losing its replication ability (Sondi & Salopek-Sondi, 2004).

3.2 UV-visible spectroscopy analysis

Figure 2 shows UV-vis spectrum of the synthesized silver nano particles. The spectrum was obtained after 48 h of mixing AgNO3 aqueous solution and balsam plant extract. The nearly colorless solution turned from faint light to yellowish and finally in brown color due to excitation of surface plasmonic nature in AgNps (Figure 3), which indicated the formation of the nanoparticles in the solution

(Pourmortazavi et al., 2015). Figure 3 Digital images of balsam plant extract (a) and the synthesized silver nanoparticles (b, c) In fact, strong absorbance occurred between the shapes of the surface plasmonic resonances (SPR) and wavelength, which related to the particle size and relative dimensions, consequently increasing in contained few nanometers sized particles. However, most of the particle size makes the SPR peak move to longer wavelength. This particles were of smaller dimensions. Figure 4c also shows enlarge observation was in line with other reports in the literature (Sondi & view of high magnification (X 15000 magnification), this revealed the Salopek-Sondi, 2004). A single broad peak at λmax = 435 nm was presence of some viburnum leaf like structures in the sample at observed in a UV-vis spectrum. The peak was correlated to the different locations. Final figure (Figure 4d) shows the FESEM image plasmonic nature of the particles as reported by earlier researchers at highest magnification (X 60000), analysis of image (Figure 4d) (Bello et al., 2017a). It has been reported by Bello et al. (2017a) that showed the presence of ~70-115nm size AgNPs. These observations the peak positions in the UV-vis spectrum of smaller particles (20nm are in agreement with the UV-vis spectrum datum, which indicated or below size) can be observed in the range of 400-420nm. The that the particles were of larger dimensions than 35nm. These finding existence of peak at 435nm in this study reveals that the nanoparticles corroborate the results obtained by Chauhan et al. (2013) who were larger than the 30nm size. reported the size of biologically synthesized Ag nanoparticles in the range of 20-70 nm. 3.3 Scanning Electron Micrsocopy 3.4 Elemental Analysis Figure 4a represents a typical overview of the sample at very low magnification (X 7500). Analysis of figure reveals the presence of The elemental analysis data of the Ag nanoparticles have been various size particles. Figure 4b is an enlarged view of synthesized represented in Figure 5. Further, EDX spectrum of these data have AgNPs (X 15000 magnification). This image reveals that sample been shown in Figure 5a, there are four peaks corresponding to the

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305 Bassam Oudh Al johny

Figure 4 FESEM analysis of the Ag nanoparticles: (a)FESEM image(s) for the surface morphology of the Ag nanoparticles at X 7500, (b) X15000, (c) X15000 at different location and (d) high magnification of x 60000. silver, zinc, carbon and oxygen. Obviously, the Ag (a) signals were due to the Ag nanoparticles in the sample. The other elements in the sample might be due to the plant source. The total elemental composition of the other elements like carbon, oxygen and zinc in the form of element weight (%) was 5.17, 3.70 and 5.21 (total

14.07%) and atomic 27.99, 15.03 and 5.18 (total 48.2%), respectively as shown in Figure 5b. It was found that the most of the samples was composed of the Ag nanoparticles, as its weight percentage was highest (85.93%). Indeed, these results revealed strong signal in the silver region and confirmed the formation of AgNps.

3.5 Functional Groups analysis (b) The FTIR analysis of Ag Nps is shown in Figure 6. Since, the AgNPs were prepared using balsam plant aqueous extract; the organic compounds might be present because of the composition of balsam plant extract. This indicates that the plant extract has played dual role in synthesizing the AgNPs. First, it played a vital role in reducing Ag1+ ions and secondly it worked as capping agent for the synthesized AgNPs. A band of 3789cm-1 is Figure 5 EDX spectrum (a) and composition of the sample (b)

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Efficacy of silver nanoparticles synthesized on Commiphora gileadensis extract against infectious bacteria 306

Acknowledgements

The author would like to thanks Dr.Yasir Anwar from biology department and Mr.M. Aldhahri, technical staff of the biochemistry department for their kindness.

Disclosure of conflicting interests

The authors declare that there is no conflict of interest to disclose.

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Adebayo HA, Song FH, Liu XT, Dai QH, Huang P, Zhang JY, Figure 6 FTIR spectra analysis of AgNps. Zhang LX (2014) Citrullus Ianatus Extract Reverses Oxidative and Haematological Dysfuntion in Carbon Tetrachloride Induced Liver Damaged Rats. International Journal of Pharmacology due to the O-H stretching between oxygen and hydrogen that is 10:218–224. attributed to water molecule. Since the sample was dried prior to the FTIR analysis, this peak intensity was lower. However, slight Ahmed MS, Kamal T, Khan SA, Anwar Y, Saeed MT, Asiri AM, moisture might have resulted in the appearance of this peak. The Khan SB (2016a) Assessment of Anti-bacterial Ni-Al/chitosan peak at 2921cm-1 indicates C-H and peak at 2300cm-1 can be Composite Spheres for Adsorption Assisted Photo-Degradation of assigned to alkynes group. The band at 1700cm-1 corresponds to Organic Pollutants. Current Nanoscience 12:569–575. C=O. The peaks observed during our study corresponds to terpenoids and flavonoid that are normally present on plant Ahmed S, Ahmad M, Swami BL, Ikram S (2016b) A review on material (Banerjee et al., 2014).Results of present study are in plants extract mediated synthesis of silver nanoparticles for agreement with the findings of previous researchers (Banerjee et antimicrobial applications: A green expertise. Journal of Advance al., 2014; Pourmortazavi et al., 2015). Results of FT–IR could be Research 7:17–28. concluded that plant contains some bioactive compounds that help Ali F, Khan SB, Kamal T, Alamry KA,Asiri AM, Sobahi TRA in both coating/capping on the nanoparticles. (2017a) Chitosan coated cotton cloth supported zero-valent nanoparticles: Simple but economically viable, efficient and easily Conclusion retrievable catalysts. Scientific Report 7:16957.

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This study elaborated the antibacterial properties of silver Ali, F, Khan SB, Kamal T, Alamry KA, Bakhsh EM, Asiri AM, nanoparticles against infectious bacteria. It may be beneficial for Sobahi TRA (2018b) Synthesis and characterization of metal researcher in order to investigate the mechanism of silver nanoparticles templated chitosan-SiO2 catalyst for the reduction nanoparticle bactericidal activity. of nitrophenols and dyes. Carbohydrate Polymers 192:217–230.

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Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 308 – 315

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

BIODEGRADATION OF THE AZO DYE (REMAZOL BLACK B) BY Pseudomonas putida ISOLATED FROM TEXTILE WASTEWATER SAMPLE

Samyah D. Jastaniah

Biology Department, Faculty of Science, King Abdulaziz University, Saudi Arabia

Received – April 03, 2019; Revision – May 21, 2019; Accepted – June 01, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).308.315

KEYWORDS ABSTRACT

Azo dye The synthetic aromatic compounds, Azo dyes, which have one or more –N=N– groups, are the most important and largest class of the used dyes with commercial applications especially in food and textile Degradation industries. The cleaning of textile wastewater containing dyes has attracted much attention in the recent Textile dyes years. The present study is an attempt to determine the ability of some bacterial isolates obtained from textile wastewater sample from Al Khumra station for decolorization of the Azo dye, Remazol black B. Azoreductase Three bacterial isolates were isolated from wastewater of textile effluents containing azo dye on basal nutrient medium. These isolated bacterial isolates were identified as Bacillus cereus, B. pumilus and Peroxidase Pseudomonas putida by phylogenetic analysis of their 16S rDNA. The most promising bacterial isolate was Pseudomonas putida and was used for further study. Results of study revealed that P. putida have Bioremediation Laccase ability of complete biodegradation (100%), if concentration is at 400 ppm while this degradation was 60 and 30 percent at 600 and 700 ppm concentration of azo dye respectively. Further, rate of biodegradation also depend on the pH and temperature of the surrounding medium, at 400 ppm concentration of azo dye, maximum degradation was obtained at pH 6.5 and temperature 40°C. This decolorization potential increased the applicability of this bacterium for the dye removal and the results suggest that the obtained isolate of P. putida can be used as a useful tool to treat waste water containing Azo dyes. Degradation potential of the bacterium depend on the presence of azoreductases, laccases and peroxidases enzymes.

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Biodegradation of the Azo dye (Remazol Black B) by Pseudomonas putida 309

1 Introduction enzymes such as laccase, azo reductase, peroxidase, and

hydrogenase (Khalid et al., 2008; Sudha et al., 2014); these enzymes The synthetic Azo dyes are most important class of dye, are degrade complex azo dyes in simpler compounds under both widely used as colorants in the textile and food and aerobic and anaerobic conditions and provide energy to these pharmaceutical industries. These azo pigments are non metalized organisms for their metabolism (Lodato et al., 2007; Sudha et al., and metalized colorants; make up 50-60% of all dyes used by the 2014). Among the identified microorganisms, due to rapid and easy world colorant industries (Anon, 1996; Aspland, 1997; Corbett, growth, survival under both aerobic and anaerobic conditions, 2000, Lalnunhlimi & Krishnaswamy, 2016). Some of the Azo bacteria is most commonly used for the treatment of azo dyes dyes are water soluble while rest are water insoluble but solvent containing wastewater. Zhang et al. (2010) detected the presence of soluble. Azo compounds are water-soluble compounds possessing Klebsiella pneumoniae, Aeromonas hydrophila, Enterobacter the characteristic –N=N- (azo) bond (Figure 1). Water-soluble azo cancerogenus, Proteus hauseri and Acinetobacter johnsonii as dyes are very important class of dyes for dyeing hydrophilic decolorizers in dye-bearing wastewater from Taiwan. Similarly, textiles such as cotton and viscose rayon. Both water soluble and Shah et al. (2013) isolated different bacterial isolates having azo dye insoluble class of azo dyes have carcinogenic properties and when degrading capability from textile industry wastewater sample. Like these dyes are absorbed by human body can undergo this, Hussain et al. (2013) isolated a dye degrading bacterial strain decomposition and form amines by the enzymatic activity of from a textile industry wastewater of Faisalabad, Pakistan in liquid body. Currently this includes the 24 amines classified as mineral salts medium. Najme et al. (2015) isolated 49 bacterial substances known to be human carcinogens (Waring, 1984; isolates from industrial waste water which can decolorize various Gregory, 1986; EPA, 1999; Ziarani et al., 2018). azo dyes like Reactive Orange 16, Reactive Yellow 2 and Reactive Red 120 under the presence of different heavy metals (Zn, Cd, Cr, Discharge of untreated Azo dyes contaminated wastewater into Pb and Cu). Among the isolated bacterial strains, Serratia sp. RN34 had highest ability to decolorize various reactive azo dyes (Yellow- 2 dye, Red-120 and Orange-16) under static conditions. Bacterial azo dying degrading capacity also depends on the pH and temperature of wastewater or surrounding mediums. Under static conditions, highest decolorization activity was reported at pH 8 and 25°C when yeast extract used as an additional carbon source. The

Figure 1 the chemical structure of Vinyl Sulfone dye, aim of the present study was isolation and identification of some also known as Remazol active and local bacterial isolates capable to degrade azo dyes from

wastewater treatment plants located at Jeddah, Saudi Arabia. environment cause considerable environmental pollution. Various 2 Materials and Methods procedures such as filtration, carbon activated, coagulation and chemical flocculation have been widely used to treat dye 2.1 Wastewater sample containing wastewater. Many textile industries have coagulation– flocculation and advanced oxidation like azo dye containing The effluent wastewater sample (50 ml) from wastewater wastewater treatment system but these procedures are complex treatment plant, WWTP at Al Khumra in the industrial area, south and costly and because of these reasons these methods are not part of Jeddah, was collected in sterile plastic bottle and feasible for many countries (Erkurt, 2010; Sivakumar et al., transferred directly to laboratory in ice bag for chemical and 2013). Furthermore, concentrations of organic matter and biological analysis. The pH and temperature of collected waste particular persistent colorants (dyes) are also affecting the water were recorded by standard procedures at sampling site. efficiency of treatment procedure (Olejnik & Wojciechowski, Different water quality parameters such as Biological Oxidation 2012). Kadlec & Wallace (2009) designed vertical-flow wetlands Demand (BOD), Chemical Oxygen Demand (COD) and Total ecosystems to remove pollutants from wastewater; this system Suspended Solids (TSS) in addition to Total Dissolved Solids imitates the natural wetlands by relying on heterotrophic (TDS) were determined by standard protocol (Rainwater & microorganisms, aquatic plants, and a combination of naturally Thatcher, 1960; USEPA, 1985; APHA, 1992; Rabah et al., 2007). occurring processes. The quantity of sodium in water is known as Sodium adsorption ratio and is measured from the equation given by Lesch & Suarez A wide range of microorganism including actinomycetes, bacteria, (1999). fungi and yeast have capability to degrade azo dyes in ecofriendly manner (Kirby et al., 2000; Olukanni et al., 2006). These 퐍퐚+ 퐒퐀퐑 = microorganisms’ posses various types of azo dyes degrading ퟎ. ퟓ(퐂퐚ퟐ+ + 퐌퐠ퟐ+)

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310 Samyah D. Jastaniah

2.2 Preparation Azo dye stock solution 2.6 Bacterial growth at different dye concentrations

Vinyl Sulfone dye (trademarked name Remazol® dye To examine the effect of different dye concentration on bacterial 6 “Remazol Black B”) is a fiber reactive dye used for silk, wool growth, 2 ml (4x10 CFU/ml) of best isolate was inoculated in a and cotton coloring, obtained from local market, Jeddah, Saudi series of parallel flasks having MSM media supplemented with Arabia. Stock solution of dye was prepared in de-ionized increased concentrations (100-1000 ppm) of Remazol Black B, water (1000 mg/l) and filter sterilized using 0.45 μm filters these culture flasks were incubated at 30°C under static conditions. and used for preparations of different concentration of the dye. For estimation of percent degradation and bacterial growth, samples The calculated volume of stock dye solution was taken, filter were collected for 3 days at each 12 hrs intervals and the sterilized and added to the sterilized medium. concentration of toxic dye and bacterial growth was estimated as mentioned earlier, uninoculated samples were used as control. 2.3 Isolation of bacteria 2.7 Factors affecting decolorization by bacteria About 5 ml of wastewater was added into 250 ml Erlenmeyer flask containing 50 ml of the Mineral Salt broth medium 2.7.1 Effect of pH on dye decolorization (MSM). The basic composition of MSM broth was: NaCl For estimating effect of pH on dye decolorization, sterile mineral (1g/l), CaCl2.2H2O (0.1g/l), MgSO4.7H2O (0.5g/l), KH2PO4 salt broth medium with 400 ppm of Remazol Black B at different (1g/l) and Na2HPO4 (1g/l). Medium was also supplemented by Remazol Black B (100 mg/l) and 0.5 (g/l) yeast extract. All pH values (4, 5, 6, 6.5, 7, and 8) was prepared in 250 ml 6 flasks having medium along with wastewater were incubated at Erlenmeyer flasks and 2 ml of the preculture (4x10 CFU/ml) was 30°C under static conditions. After 3 days, bacterial cells were inoculated in each flask, each treatment was replicated thrice. collected by centrifugation at 5000 rpm and these were Uninoculated medium was used as control. All the inoculated and transferred to the fresh medium containing flasks with Remazol uninoculated flasks were incubated at 30°C under static Black B (100 mg/l). After 48 hrs of incubation bacterial growth conditions. The percentage decolorization was measured as was estimated and this was followed by the application of 1.0 mentioned earlier. ml culture broth on Nutrient Agar containing Remazol Black 2.7.2 Effect of temperature on dye decolorization: (100 mg/l). The colonies exhibiting color removing ability (with clear zones around) were selected, purified and preserved on Erlenmeyer flasks containing 50 ml mineral salt broth medium at Nutrient Agar slant at 4°C for further studies. Concerning pH 6.5 were used to determine the best temperature for dye Escherichia coli, it was obtained from King Fahad General degradation. The used concentration of the dye was 400 mg/l and Hospital, Jeddah, Saudi Arabia all flasks was inoculated with 2 ml of the preculture (4x106 CFU/ml) and were incubated at 25, 30, 35, 40, 45 and 55°C for 3 2.4 Preculture of the bacterial isolates days under static condition. Uninoculated medium was used as A loopfull of culture of the selected bacteria were inoculated in control, percentage decolorization was measured at each 12 hours 250 ml Erlenmeyer flasks containing 50 ml of Nutrient broth intervals and obtained results were compared with controls. medium for 2 days at 30°C and 120 rpm. 2.8 Bacterial identification 2.5 Selection of the most active bacterial isolates The bacterial isolate which showing highest dye degradation ability For bacterial selection, the isolates which can tolerate higher was identified by using morphological, physiological, biochemical concentration of the Azo dye, were selected and maintain on and molecular studies. Some morphological and physiological MSM liquid medium supplemented with Remazol Black B (100 characters were determined as per the Bergey’s manual of mg/l) and yeast extract (0.5 g/l). Each flask was inoculated with determinative bacteriology. Biochemical tests like Methyl Red, 2 ml of bacterial preculture (4x106CFU/ml). At every 12 hrs Voges-Proskauer, Indole, Citrate, Catalase, and Oxidase, Nitrate intervals, bacterial growth was measured by turbidity (A450 nm) reduction and hydrolysis of Casein, Starch, Urea and Gelatin were and the culture broth was centrifuged at 5,000 rpm for 15 also determined. Utilization of different carbon sources, D-glucose, minutes. Percent decolorization was monitored by measuring D-fructose, galactose, mannitol and D-maltose and some nitrogen initial and final absorbance at 595 nm and calculated according sources were detected at 30°C for 24-48 hrs. to the equation given by Saratale et al. (2009). Crude lysate of cells were prepared, DNA was extracted and Initial OD −Final OD Percent Decolorization = × 100 subjected to PCR amplification by using the 16S rRNA forward Initial OD

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Biodegradation of the Azo dye (Remazol Black B) by Pseudomonas putida 311

primer (5′- AGTTTGATCATGGTCAG-3′) and reverse primer color of the collected water was dark gray, it was slightly

(5′-GGTTACCTTGTTACGACT-3’). The primers were designed acidic (pH 6.45) and have mild temperature. In general, the based on the highly conserved region of 16S rRNA from various obtained pH value fall within the limit (pH 7.0 to 8.5) bacteria (Weisberg et al., 1991). The amplified PCR product was suggested by the World Health Organization (WHO, 1989; sequenced using a big dye terminator cycle sequence kit. The DWAF, 1996). Results of present study are in agreement with product of the sequencing reaction was analyzed using DNA the findings of Tyagi & Mehra (1990) while these are Sequencer ABI PRISM 310 genetic analyzer (Perkin Elmer) and contradictory to the findings of Shah (2013) those who DNA sequence was compared with sequences of some members reported alkaline nature (pH 7.45) of waste water, this of the genus available through GenBank database. difference might be because of the nature of pollutant in wastewater. The pH value of water is very important, as it 2.9 Statistical analysis affects the development of microbial community and other All decolorization experiments were performed in three sets and biochemical reactions. Contamination of water by textile mean values were calculated. pollutant also affects the solubility of gases in water which marked by higher BOD, this higher BOD value indicate the 3 Results and Discussion presence of pollutant and higher microbial load in tested water samples (APHA, 1992). Further, electrical conductivity of the Jeddah Bay received massive discharges of partly-treated collected water samples was reported 3.3 dS/m while mean wastewater, Al Khumra which is a multiport diffuser pipe also COD value was reported 17.32 mg/l. On the other hand, contribute in enhancing waste water level of bay (Peña-García et biochemical oxygen demand (BOD) of the collected al., 2014). Textile dyeing, leather tanning, paper and sugarcane wastewater sample was reported 3.48 mg/l and the value of industries produce large quantities of wastewater which cause total suspended solids (TSS) were found 1134.9 mg/l. The serious water pollution not only in rivers, pond or bay but also + - 3- concentrations of NH4 , NO3 and P were found 36.31, 21.48 contaminate groundwater and soil (Abrahart, 1977; Rafi & and 4.56, mg/l, respectively (Table 1). In the present study, Daugherty, 1997; Senan & Abraham, 2004; Akarslana & BOD value was reported lower than the permissible limits. Demiralay, 2015). In current study physico-chemical properties of Lokhande et al. (2011) reported higher BOD values (1047.3 the collected textile waste effluent were estimated (Table 1). The mg/L) for the water sample collected from pharmaceutical

Table 1 Chemical and biological analysis of industries while it was reported 776.2 mg/l and 535.8 mg/L the collected wastewater for the samples collected from dyes and paint industries. In

Parameter Wastewater FAO present study electrical conductivity was reported higher than that recommended concentration (0-3.0) of FAO (1985), pH 6.45 6-9 which means collected water samples are not good for general Temperature 43ᵒC - use (Abdo et al., 2010). It was well known that wastewater - Color Gray - contained many nutrients and ions especially nitrate (NO 3 ), affecting the plant growth adversely (Magesan, 2001). In EC (dS/m) 3.30 0-3 present study, sodium adsorption ratio (SAR) was reported + NH4 (mg/l) 36.31 30 6.60 which is very higher the recommended limit and it - NO3 (mg/l) 21.48 - indicate the unsuitability of this water for irrigation and cultivation purpose. Further, TDS is high for the collected P3- (mg/l) 4.56 - samples which reduce purification of wastewater by SAR 6.60 15 microorganisms (Jiunkins, 1982; Kim, 1994). TDS (mg/l) 1735.40 2000 The water samples collected from wastewater treatment plant TSS (mg/l) 1134.90 - of Al Khumra, industrial area, were spread on mineral salt COD (mg/l) 17.32 - broth medium containing 100 ppm of the Remazol dye. BOD (mg/l) 3.48 - Bacterial colonies surrounded by clear zone were consider as an effective strain and were selected for further study related TS :Total Solids, TDS: Total Dissolved Solids, TSS: Total Suspended Solids, EC = Electrical Conductivity (dS/m), COD: Chemical Oxygen to the azo dye removal capability; total three bacterial isolates Demand (mg/l) , BOD: Biological Oxygen Demand (mg/l), FAO: were selected and isolated. These isolated isolates Food and Agriculture Organization (1985), -: not detected were characterized and identified according to Bergey’s Manual of

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Systematic Bacteriology, physiological and biochemical tests

(Holt et al., 1993; Harrigan, 1998), these isolated strains were identified as Bacillus pumils ABO23, Bacillus cereus ABO13 and Pseudomonas putida ABO23. Among these, Bacillus pumils ABO23, B. cereus ABO13 were Gram positive spore forming bacteria while P. putida ABO23 was gram negative, rod shape, non spore forming bacterium. Identification was confirmed by using 16 rRNA studies and phylogenic trees of the selected bacteria compared with available genera database (Figures 2 and 3). Figure 4 showed the percentages of decolorization by the selected three most promising bacteria isolates, grown on broth medium supplemented with 100 ppm Figure 2 Phylogenic relationship between Bacillus pumils of Remazol B for 3 days, and these results were compared ABO23, B. cereus ABO13 and other related reference isolates with E. coli. Further, the decolorization percentages after 72 based on the 16S rRNA gene sequence analysis hours were 100, 80 and 72 percent for P. putida ABO23, Bacillus cereus ABO13 and B. pumils ABO23, respectively decolorization percentage was reported at pH 6.5 but this dye while it was less than 20% in case of E. coli. Figure 5 degradation percentage reduced at pH 6 and 7 and the lowest represents the percentage decolorization by the P. putida percentages were at pH 4, 5 and 8 (Figure 6). Findings of ABO23 grown on broth medium containing increasing present study are in agreement with the findings of Chang et concentrations of Remazol black B for 3 days. The percentage al. (2001) and Shah et al. (2013). Similarly, Aburas (2016) of degradation was 100 percent up to 400 ppm concentration isolated P. putida from waste water sample and reported of Remazol black B and afterword increasing concentration of highest ability of PHB production. Azo dye degradation was Remazol black B decreased degradation. At 700 ppm, enhanced by alkaliphilic bacterial consortium (Mahmoud decolorization percentage was less than 30 percent after 72 hrs et al., 2017) or by special fungal degradation including of inoculation (Figure 5). Effect of pH and temperature was Aspergillus niger strain isolated from textile wastewater. also evaluated in present study and maximum decolorization Bioremediations of contaminants and toxins using bacteria has percentage was recorded at pH 6.5 and temperature 40°C been intensified and reported by many authors (Dawkar et al., whereas 100% decolorization was found after 60 hrs instead of 2008; Singh et al., 2014). Similarly, Roy et al. (2018) reported 72 hr at pH 7 and 30°C (Figure 6 & 7). The pH value of the that bioremediation of industrial effluent containing medium affect degradation of the dye and maximum textile dyes using bacteria is an emerging and important tool in

Figure 3 Phylogenic relationship between Pseudomonas putida and other related reference isolates based on the 16S rRNA gene sequence analysis

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Biodegradation of the Azo dye (Remazol Black B) by Pseudomonas putida 313

120 120 Ps. putida 100 100 100 ppm B. pumilus 80 80 200 ppm 60 B. cerrus 60 400 ppm 40 40 600 ppm E. coli

20 20 700 ppm

% of decoloration decoloration of % % of decoloration decoloration of % 0 0 12 24 36 48 60 72 84 12 24 36 48 60 72 84

Time in hours Time in hours

Figure 4 Percent decolorization of the three bacteria isolates grown Figure 5 Percent decolorization by the selected P. putida ABO23, on broth medium supplemented with Remazol B for 3 days and grown for 3 days on broth medium containing increasing results compared with E. coli concentrations of Remazol B

120 120

100 25°C 100 pH4 80 30°C 80 pH5 35°C pH6 60 60 40°C pH6.5

decoloration 40 45°C 40 pH7

55°C 20 pH8 % of of %

20 decoloration of % 0 0 12 24 36 48 60 72 84 12 24 36 48 60 72 84

Time in hours Time in hours

Figure 6 Effect of temperature on degradation of Remazol Black B Figure 7 Effect of pH on degradation of Remazol Black B by the by the selected P. putida ABO23 selected P. putida ABO23

environmental protection and pollution treatment. In and antibiotic susceptibility of pathogenic bacteria of Ismailia conclusion, industrial effluent containing textile dyes was canal water, River Nile, Egypt. Journal of American Science 6: considered as mutagenic and mitotic poisoning water and 234-250. bioremediation of these toxic materials by bacteria, is Abrahart EN (1977) Dyes and their Intermediates. New York: becoming an emerging and important technique. All three Chemical Publishing, Pp. 1–12. selected bacterial isolates from the textile effluent showed excellent decomposition and decolorization of the textile Azo Aburas MMA (2016) Production of poly β hydroxybuthyrate from dye. Among these P. putida reported most potent one under Pseudomonas putida MAO12 isolated from wastewater sample. the best conditions for bioremediation process. Journal of American Science 12 :107-112.

Conflict of interests Akarslana F, Demiralay H (2015) Effects of textile materials harmful to human health. Acta Physica Polonica A No. 2-B Vol. There is no conflict of interest. 128: 407-408.

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Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 316 – 328

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

DOCUMENTATION OF PLANT DIVERSITY IN MARKETS OF GOALPARA DISTRICT OF ASSAM USED FOR FOOD AND GENERAL HEALTHCARE

1 1 2 2 Kaustav Kalyan Sharma , Vivek Ghose , Dipamoni Nath , Dulal Chandra Boruah , Dharmeswar Barman2, Sheemanta Jyoti Deka1, Someswar Rao1,2*

1Advanced level Institutional Biotech Hub, Department of Botany, Goalpara College, Goalpara, Assam, India. 2Department of Botany, Goalpara College, Goalpara, Assam, India

Received – March 27, 2019; Revision – May 26, 2019; Accepted – June 07, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).316.328

KEYWORDS ABSTRACT

Goalpara district Since the dawn of civilization public markets have played an important role in supplying goods for maintaining health and nutrition of mankind, especially in the rural areas. The present study was Weekly markets designed to document the local plants and plant products that are sold in different rural weekly markets Plants of Goalpara district of Assam, India, which served particularly as food supplements or ingredients for local cuisine. This survey was conducted across a period of one year following a standard questionnaire, Cultural beliefs personal interviews with the sellers, documenting the plants sold, their parts used, mode of use and price range. Results showed that 71 number of plant species belonging to 41 families are being used as food Nutrition and to treat various common ailments for maintaining overall health. Araceae and Rutaceae were found to be the most dominant plant families followed by Euphorbiaceae, Amaranthaceae, Apiaceae, Malvaceae, Rubiaceae and others as per the number of plant species is concerned The plant products constitute fruits, flowers, leaves, stems, tubers and rhizomes. The study gives an idea of the plant diversity of the area along with glimpses of the prevailing cultural beliefs and socio-economic condition of the locals.

* Corresponding author All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a E-mail: [email protected] [email protected] (Someswar Rao) ; Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All rights reserved.

Documentation of Plant diversity in markets of Goalpara district of Assam 317

1 Introduction of the district is 755133 in 2011 (Deka, 2016). It is quite

common that different ethnic groups of the tribal dominated Human dependence on plants for their basic requirements like Goalpara district largely depends on plant and their derivatives food, medicine, clothes and shelter is as old as mankind itself for curing various health disorders. Herbaceous plants play a and still in the modern age the majority of commercial major role in disease cure and control while extracts derived products including pharmaceutical and healthcare, food and from various parts of shrubs and trees are also deployed for beverages, textiles, cosmetics and aromas are obtained from treatment. The use of roots, stem, leaves, bark, seeds or even plants (Goyal et al., 2007; Khanuja, 2012; Gupta et al., 2018). the whole plant is quite common. These plants are either All around the globe, traditional markets are considered as collected from nearby forest areas or sometimes cultivated in places for trade and marketing of plant and plant products, the homesteads or home gardens. It is worth mentioning that where cultures are expressed by means of regional trade, different kinds of weeds growing in the agricultural fields as likewise markets acts as meeting spots where various well as in the vicinity of the residences are also used for ecologically and culturally diverse communities display and disease prevention and cure (Deka & Nath, 2014). The sell a diverse array of minerals, animals, plants and their dependence on plant and plant based products as sources of derivatives. (Hooper et al., 1937; Linares & Bye, 1987; medicine and primary health supplements is still prevailing in Nicholson & Arzeni, 1993; Martin, 1995; Cunningham, 2001; these areas largely because of the prevailing traditional beliefs Parente & da Rosa, 2001; Hanlidou et al., 2004; Williams et and ancestral cultures, easy access and cost effective nature of al., 2007a; Williams et al., 2007b; Williams et al., 2009; the products. In this aspect, the markets play a vital role as Giraldo et al., 2009; Silalahi et al., 2015; Tinitana et al., 2016; these are very well organized, generally established in the Tibuhwa, 2018). Indigenous people living on their traditional same day in every week and concentrate on collection, territory largely rely on plants and plant products sold in the accumulation and selling of different products from far public markets for general healthcare. This reliance can be reaching areas in the same spot. The present study aims to attributed towards the cost-effective and easy accessible investigate the diversity of edible plants and plant products nature of the products. In general, the vendors in the market sold in different local markets of Goalpara district, Assam and that sell plants for therapeutic and nutritional purposes acts as to understand their utilization by the locals as food and in popular prescribers, conducting a practical exhibition and prevention, treatment and healing of diseases as well as explanation of empirical knowledge about indications, improvement of general healthcare. preparation methods and use, plant parts and quantities used and other pertinent information (Santos et al., 2009; Silva & 2 Material and Methods Peixoto 2009; Leitao et al., 2009; De Carvalho et al., 2015). 2.1 Study area Situated on the western part of Assam, Goalpara district shares its boundary with West and East Garo hills district of The geographical location of Goalpara district is between 25° Meghalaya on the south, while the river Brahmaputra flows all 53' N to 26° 30' N latitude and 90° 07' E to 91° 05' E longitude along the north. Kamrup and Dhubri districts of Assam are (Rabha 2014). The area of Goalpara district is 1824 sq.km and located in the east and west respectively (Rabha, 2014). Semi the total forest area is 36915.27 hectares (Rabha, 2012). There evergreen and mixed deciduous are the main vegetation types are five revenue circles in the district viz., Lakhipur, Balijana, of the area while some other subtypes also prevails. Taking Rangjuli, Dudhnoi and Matia while Jaleswar, Lakhipur, account of the immense floristic diversity, Goalpara can be Kharmuja, Balijana, Krishnai, Matia, Kushdhawa and Dudhnoi considered a rich district of Assam (Deka & Nath, 2014). comprise the eight blocks of the district (Deka, 2016). The survey was conducted in different weekly markets of Goalpara Goalpara district is inhabited by many tribes and communities. district viz., Dhupdhara, Rangjuli, Darangiri, Dhanubhanga, Muslims community and tribes such as Rabha, Koch and Garo Dhudhnoi, Krishnai, Tukura, Makri, Agia and Lakhipur over a comprises the main population of the district while other tribes period of 12 months in respective days of the weeks when the and communities like Bodo, Kalita and Hindu Bengalis are markets were held. Location map of the study area of Goalpara also among the residents (Deka & Nath, 2014). The population District of Assam, India is presented in Figure 1.

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Figure 1: Location map of the study area of Goalpara District in Assam, India. (Map Source - Assam District Factbook 2018).

2.2 Data Collection: various local markets in Goalpara district of Assam. The results represented 71 plant species belonging to 41 Families which have Data collection was carried out by following a standard been traditionally used by the locals for maintaining health and questionnaire which includes the local name, type of plant, plant nutrition. In case of family, Araceae and Rutaceae were found to part sold, mode of use, price range, place of origin of the plant, whether cultivated or collected from wild etc. The sellers were be the most dominant plant families followed by Euphorbiaceae, casually interviewed of their products and photographs were taken Amaranthaceae, Rubiaceae, Malvaceae and others as per the with due permission. Emphasis was given on selecting and number of plant species per family is concerned. The plants documenting the plants that are not so commonly found. Later the include all forms of vegetation viz., herbs, shrubs, trees, climbers collected documents were compared with standard literatures and aquatic vegetation (Figure 2). Different parts of the plants (Patiri & Borah, 2007) to know their scientific names and other were in use (Figure 3). Table 1 represent the various plants sold in parameter such as family name, vernacular names, growth different markets of Goalpara district, Assam, along with their characteristics etc. families, local Assamese names, growth habits, parts used, their

3 Results uses and sold prices. The numbers of plant species belonging to different plant families are given in Figure 4. Figure 5 and Figure 6 The present study was conducted to document the diversity of shows the local products in different form available at different agricultural and non agricultural plant and plant products sold at markets from Goalpara District.

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Documentation of Plant diversity in markets of Goalpara district of Assam 319

Table 1: Plants sold in markets of Goalpara district, Assam with their family, local names, growth habit, parts used/ uses and price.

Botanical name with Family Local Name/ Growth Parts used/ Uses Price Vernacular habit name Bel T Aegle marmelos (L.) Correa. (Rutaceae) Fruits are used to prepare juice. Rs.5/piece. Alocasia acuminata Schott. (Araceae) Kochu H Tubers are used as vegetables. Rs. 30-40/kg.

Boga kochu H Leaves, stem and tubers are eaten, Alocasia cucullata (Lour.) Schott. (Araceae) leaves used as to cure external body Rs.5/bundle. pain. Alocasia indica (Lour.) Koch. (Araceae) Man Kochu H Tubers/ roots are used as vegetables. Rs.30-40/kg.

Alocasia odora (Lindl.) K.Koch. (Araceae) Dudh Kochu H Petioles are used as vegetables. Rs.5/bundle.

Amaranthus virdis L. (Amaranthaceae) Khutura sak H Leaves are used as vegetables. Rs.10/ 3-4 bundles.

Amaranthus spinosus L. (Amaranthaceae) Katakhutura H Leaves are used as vegetables Rs.10/ 3-4 bundles.

Amaranthus tricolor L. (Amaranthaceae) Datha H Leaves are used as vegetables Rs.10/ 4-5 bundles.

Artocarpus heterophyllus Lamk. (Moraceae) Kothal T Fruits are eaten raw. Rs/piece (price vary on size). Kordoi T Fruits are eaten raw also used to Averrhoa carambola L. (Oxalidaceae) Rs. 20-30/kg. make curry. Maha-neem T Leaves are eaten for bitter taste, used Azadiracta indica A. Juss. (Meliaceae) to treat fever, worm infection and Rs. 5/bundle. skin diseases. Baccaurea ramiflora Lour. (Euphorbiaceae) Leteku T Fruits are eaten raw. Rs. 10/bundle.

Bacopa monneri (L.) Pennell (Scorphulariaceae) Brahmi sak H Leaves are used as vegetables Rs. 5/bundle.

Ronga Puroi/ Pui C Basella alba L. var. rubra (L.) Stewart. (Basellaceae) Leaves are used as vegetables Rs. 5/bundle. xak Basella alba L. var. alba (Basellaceae) Boga Puroi sak C Leaves are used as vegetables Rs. 5/bundle.

Brassica nigra (L.) W.D.J. Koch. (Brassicaceae) Soriyoh Sak H Leaves are used as vegetables Rs.10/ 4-5 bundles.

Canavalia cathartica Thouars (Fabaceae) Kamtal urahi C Fruits used as vegetables. Rs. 50/ kg.

Capsicum chinense Jacq. (Solanaceae) Bhot Jolokia S Fruits are eaten raw. Rs. 10/3-4 pieces. Amita T Ripe fruits are eaten raw. Unripe Rs. 40/kg (ripe fruit) Carica papaya L. (Caricaceae) fruits are used to treat stomach Rs. 15-20/Kg (raw fruit) problems. Flowers used to treat fever. Rs. 10/part (flowers) Bor manimuni H Leaves are used to treat stomach Centella asiatica (L.) Urban ( Apiaceae) Rs.5/ part. troubles. Chenopodium album L. (Chenopodiaceae) Bhatua/Jilmil H Leaves are used as vegetables Rs.10/ 4-5 bundles.

Cinnamomum tamala (Buch.-Ham.) Nees & Tejpat T Leaves are used as flavouring agent. Rs.5/ bundle. Eberm. (Lauraceae) Citrus grandis (L.) Osbeck (Rutaceae) Robab tenga T Fruits are eaten raw. Rs.10/piece.

Jora nemu S Fruits are eaten raw considered as a Citrus medica L. (Rutaceae) source of Vitamin C and Rs.10/2pieces. antioxidants. Kola kochu H Tubers are used as vegetables. Colocasia esculenta (L.) Schott (Araceae) Rs.5/ bundle. Leaves are used to treat joint pains. Moraphat S Tender leaves are eaten as Corchorus capsularis L. (Malvaceae) Rs.10/ 3-4 bundles. vegetables.

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Botanical name with Family Local Name/ Parts used/ Uses Price Growth Vernacular habit name Rhizomes are used as spice, against Rs. 60/kg (raw) Curcuma longa L. (Zingiberaceae) Halodhi H arthritis. Paste of rhizomes applied Rs. 250/kg(powder) topically over small burns. Fruits are eaten raw or used in pickles and to prepare curry. Fruit decoction Dillenia indica L. (Dilleniaceae) Ou tenga T Rs.5/piece. used to treat hair fall. Used to prepare fish curry. Tubers are used as vegetables. Used Dioscorea alata L. (Dioscoreaceae) Kath alu C Rs. 40-50/kg. to treat indigestion.

Dioscorea bulbifera L. (Dioscoreaceae) Gosh Alu C Tubers are used as vegetables. Rs. 30-40/kg. Diplezium esculentum (Retz.) Sw. Leaves and tender stem used as Dhekia sak H Rs. 10/ bundle. Woodsiaceae (Athyriaceae) seasonal vegetables.

Elaeocarpus floribundus Bl. (Elaeocarpaceae) Jolphai T Fruits are used in pickles, eaten raw Rs. 25-30/kg.

Leaves and tender shoots are used to Enhydra fluctuans Lour. (Asteraceae) Helochi H Rs. 10/ 2-3bundles. prepare curry in local cuisine. Leaves are used as garnishing and Eryngium foetidum L. (Apiaceae) Man dhania H Rs. 10/ bundle. flavouring agent.

Euryle ferox Salisb. (Nymphaeaceae) Makhana AV Fruits are eaten raw. Rs. 4-5/piece.

Flacourtia jangomas (Lour.) Raeusch (Salicaceae) Ponial T Fruits are eaten raw. Rs. 40/kg.

Fruits are eaten raw. Dried fruit rinds Rs. 5/piece (raw) Garcinia pedunculata Roxb. (Clusiaceae) Bor thekera T used to cure dysentery and diarrhea. Rs. 200/Kg (dried rinds). Also used to prepare fish curry. Leaves are used to prepare curry with Rs.10/bundle (leaves). Hibiscus subdarifa L. (Malvaceae) Mesta tenga H lentils. Fruits are used to prepare jam Rs.10 /part (fruits) and jelly. Leaves are used as vegetables and Houttuynia cordata Thunb. (Saururaceae) Masundari H Rs.10/bundle. used against diarrhea. Leaves paste is used to enhance Hydrocotyle sibthorpioides Lamk. (Apiaceae) Xaru manimuni H Rs. 10/ 3-5bundles. appetite after disease.

Hedyotis corymbosa (L.) Lam. (Rubiaceae) Bon jaluk H Leaves are used as vegetables. Rs.5/bundle. Leaves are used to prepare curry in Ipomoea aquatica Forsk. (Convolvulaceae) Kolmou AV local cuisine, decoction of leaves used Rs.5/2bundles. against liver problems. Tubers are eaten raw or as vegetables Ipomoea batatas (L.) Lamk. (Convolvulaceae) Mitha Alu C Rs. 30-40/kg. in local cuisine. Leaves are bitter in taste used against Leucas plukenetii (Roth.) Spreng. (Lamiaceae) Dron/Doron xak H Rs.5/bundle. jaundice, and also in local cuisine. Leaves are used as vegetables in local Malva verticillata L. (Malvaceae) Lofa sak H Rs. 10/ 3-4bundles. cuisine. Roots are used as vegetables. Used to Rs. 40-60/kg. Manihot esculenta Crantz. (Euphorbiaceae) Simalu alu S treat stomach problems. Rs. 20-30/bundle. Leaves are used as appetite enhancers Mentha virdis L. (Lamiaceae) Pudina H and as flavouring and garnishing Rs. 10/ 3-5bundles. agent. Fruits are used as vegetables. Dried Meyna spinosa Roxb. ex. Link. (Rubiaceae) Moinphal T fruits are soaked in water and used Rs.10 /part (9-10 fruits per part)

against dysentery. Fruits and leaves are used as Rs. 200/kg. (tender drumsticks). Moringa oleifera Lamk. (Moringaceae) Sajina T vegetables, leaves decoction is used Rs.60/kg. (mature drumsticks). to treat fever and diabetes.

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Botanical name with Family Local Name/ Growth Parts used/ Uses Price

Vernacular habit name

Leaves are used as flavouring agent Murrya koenigii (L.) Spreng. (Rutaceae) Narasingha S Rs. 10/ 3-4bundles. and appetizer.

Athia kol/Bhim Fruits are eaten raw, stem and flower Musa balbisiana Colla. (Musaceae) H Rs. 40-50/ dozen. kol as vegetables.

Nyctanthes arbor–tristis L. (Oleaceae) Sewali phul T Flowers are used to enhance appetite. Rs.5 /part.

Nymphaea rubra Roxb.ex Andrews (Nymphaeaceae) Seluk AV Roots are used as vegetables. Rs.40-50 /kg.

Leaves decoction is used to cure Paederia foetida L. (Rubiaceae) Bhedailota C stomach ache, prepared with fish to Rs. 10/ 2-3bundles. enhance appetite. Flowers are dried and used to treat Phlogocanthus thyrsiflorus (Roxb.) Nees (Acanthaceae) Ronga bahka tita S Rs.20 /part. jaundice and fever.

Phyllanthus acidus (L.) Skeels (Euphorbiaceae) Holfoli T Fruits are acidic in taste eaten raw. Rs.5 /part.

Fruits are considered as source of Phyllanthus emblica L. (Euphorbiaceae) Amlokhi T Vitamin C, consumed for general Rs. 30-40/kg. health improvements. Leaves are used as mouth freshener Piper betle L. (Piperaceae) Pan C after meal. Paste of leaves used to Rs. 10/Bundle treat anal fissure. Fruits are used as spice made paste Rs.20 /part (young peppercorns). Jaluk/Gol Piper nigrum L. (Piperaceae) C with honey to cure cough and sore Rs.90/100gms (dried morich throat. peppercorns).

Polygonum chinense L. (Polygonaceae) Madhusuleng H Leaves are used to treat dysentery. Rs.5 /bundle.

Fruits are used as vegetables with Solanum indicum L. (Solanaceae) Tit bhekuri S Rs.10 /part (25-30 fruits per part) rice. Leaves are used to treat wound Spilanthes paniculata Wall. ex D.C. (Asteraceae) Huhuni sak H Rs.5 /bundle. healing. Fruits are eaten raw, prepared curry Spondias pinnata (L.f.) Kurz. (Anacardiaceae) Amora T Rs 20-30 /kg. with fish.

Dried leaves are used against fever, Chirota tita H Rs.5 /dried bundle. Swertia chirayita (Roxb. ex. Flem.) Karsten stomach problems. (Gentianaceae) Fruits are eaten raw or in curry and Rs.40-60 /kg. (dried) Tamarindus indica L. (Fabaceae) Teteli T pickles for sour flavor. Rs. 10/part (raw fruits). Dried fruits are used as mouth Terminalia chebula Retz. (Combretaceae) Silikha T Rs. 10/part. fresheners, Fruits are used to cure gastritis, Terminalia bellerica (Gaertn.) Roxb. (Combretaceae) Bhomora T Rs.40-50 /kg. blotting and constipation. Trapa natans L. var. bispinosa (Roxb.) Makino Singori/Pani AV Fruits are eaten raw. Rs.50-60 /kg. (Trapaceae) phal

Zanthoxylum oxyphyllum Edgw. (Rutaceae) Mezenga S Young leaves are used as vegetables. Rs. 10/ 1-2bundles.

Rhizomes are used as spice condiments, used to treat minor Zingiber officinallis Rosc. (Zingiberaceae) Ada H stomach problems. Dried rhizomes Rs.80-100 /kg. are used as mouth fresheners and digestive agents. Fruits are eaten raw, or used in Zizyphus mauritiana Lamk. (Rhamnaceae) Bogori T Rs.30-40 /kg. pickles. Used to treat diabetes. T- tree; S- shrub; H- herb; C- climber; AV- aquatic vegetation

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Aquatic vegetations Shrubs 6% 11% Herbs 39%

Climbers 13%

Trees 31%

Figure 2 Plant growth habits.

Flowers Petioles 5% 1% Rhizomes, Roots & Tubers Fruits 13% 37%

Leaves, Stems & Shoots 44%

Figure 3 Plant parts used

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Documentation of Plant diversity in markets of Goalpara district of Assam 323

Zingiberaceae Trapaceae Solanaceae Scorphulariaceae Saururaceae Salicaceae Rutaceae Rubiaceae Rhamnaceae Polygonaceae Piperaceae Oxalidaceae Oleaceae Nymphaeaceae Musaceae Moringaceae Moraceae Meliaceae Malvaceae

Plant Families Plant Lauraceae Lamiaceae Gentianaceae Fabaceae Euphorbiaceae Elaeocarpaceae Dioscoreaceae Dilleniaceae Convolvulaceae Combretaceae Clusiaceae Chenopodiaceae Caricaceae Brassicaceae Basellaceae Athyriaceae Asteraceae Araceae Apiaceae Anacardiaceae Amaranthaceae Acanthaceae

0 1 2 3 4 5

Number of Species

Figure 4 Number of indivials from each plant families recorded in the survey.

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324 Sharma et al.

A B

C D

E F

Figure 5 Local products sold at different markets. A- Flowers of Caraya papaya. B- Flowers of Phlogocanthus thyrsiflorus. C- Fruits of Meyna spinosa. D- Fruits of Hibiscus subdarifa. E- Tubers of Nymphaea rubra. F- Leaves of Zanthoxylum oxyphyllum.

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Documentation of Plant diversity in markets of Goalpara district of Assam 325

G H

I J

K L

Figure 6 Local products sold at different markets. G- Roots of Manihot esculenta. H- Dried Swertia chirayita. I- Tubers of Colocasia esculenta. J- Local Mushroom. K and L- Women vendors at different markets.

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4 Discussion markets which give an idea about the species richness of these open air weekly markets of Goalpara district of Assam. A Rural markets represent one of the main supplementary incomes surprising fact about these open air markets is their ability to for local people that are particularly utilized at household level. attract customers from distant places by virtue of this abundance Moreover, sellers along with their own goods also sell purchased and unique species composition. These much awaited open air products in order to meet the demands of the local people (Vlkova markets act as support system for the sellers to generate et al., 2015). The sellers in the weekly markets of Goalpara financial assistance from the home grown or collected products district constitute men and women of all age groups but it was as for some it is the sole source of income and livelihood. On observed that the majority of traders were women aged between the other hand the consumers gain advantage from the fresh and of 30 – 70 years. This observation is in support of some earlier nutritive natural products sold at these markets. investigations that reported that the women constitute the major part of the traders of medicinal plant materials (Idu et al., 2010; Conclusion Vlkova et al., 2015). The study documented the current plant diversity of the various The major plant products traded in the area were derived from markets of Goalpara district of Assam, India. As many as 71 plant plants of all growth habits but trees and herbs were species being recorded, sold in the local weekly markets which predominant sources. The collected plants were found to be fetch good income to rural communities. The edible species also very common among the people and were used in traditional form a good source of nutrients in local diets besides improving healthcares for a variety of disease conditions such as fever, the overall health. The study reflects the high dependence on these jaundice and other liver problems, skin diseases, joint pain, resources by the tribal and non tribal communities of Goalpara small burns, hair fall, indigestion, dysentery, diarrhea, district. Furthermore it throws some light in identifying the diabetes, constipation, cold and cough etc. and general health fundamental plants which are being sold in these local markets, improvements (personal communications, table 1). The leafy their availability, price range with their common uses. These types vegetables, fruits and tubers also form an integral part of the of studies may be helpful in identifying rare and endemic plant regular local cuisine for the people of Goalpara district and species for their conservation and sustainable use. many are used as appetite enhancers. The rates of the sold Acknowledgement items varied from market to market, season to season depending on the quality and abundance. Occasionally locally The authors are highly grateful to the Department of grown mushrooms are also sold in the markets. Biotechnology, Govt. of India for the financial assistance. We deeply acknowledge the co-operation received from the local A number of studies have been carried out on the trade of people interviewed during out the survey. medicinal and wild edible plant from across the globe. Dogan et al. (2013) reported, a total of 46 wild edible plants sold in the Conflict of Interest local markets of Izmir, Turkey that play an important role in their diet. Plants used were either eaten raw, cooked by boiling No conflict of interest. in water, frying in oil or baked to be served as dishes such as stew, salad as hot drink. As per Dogan & Nedelcheva (2015), in References the open air markets at both sides of the Bulgarian-Turkish Assam District Factbook (2018) Goalpara District. (Key Socio- border a total of 41 wild plants belonging to 20 families are economic Data of Goalpara District, Assam) January, 2018. sold, among which approximately 17% of the plants were http://www.datanetindiaebooks.com/District_Factbook/Assam/Go common and widely reported. Kasper-Pakosz et al. (2016) alpara. pdf, accessed on 16 February 2019 . recorded 468 species of plants and 32 species of mushrooms sold in the open-air markets of southeastern Poland. Likewise, Cunningham AB (2001) Applied Ethnobotany: People Wild Plant Khruomo & Deb (2018) documented a total of 47 indigenous Use and Conservation. People and Plants Conservation. London: wild edible fruits belonging to 29 family and 39 genera from Earthscan Publications. Nagaland, India. In another study Łuczaj et al. (2013) stated that in every market in Dalmatia (southern Croatia), wild edible De Carvalho Nilo Bitu V, de Carvalho Nilo Bitu V, Matias EF, de plant are sold as plant mixes, usually composed of just a few Lima WP, da Costa Portelo A, Coutinho HD, de Menezes IR . species of wild as well as cultivated vegetables to a lesser (2015) Ethnopharmacological study of plants sold for therapeutic extent. In the present study many plants and plant products are purposes in public markets in Northeast Brazil. Journal of documented that are rarely found in the regular day to day Ethnopharmacology 172: 265–272.

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Cough or Bronchial Problems in Goalpara District (N.E. India). ethnobotany in Petropolis and Nova Friburgo (Rio de Janeiro, International Journal of Pure and Applied Bioscience 2: 80-87. Brazil). Revista Brasileira de Farmacognosia 19: 333-342.

Deka P (2016) A Study on Disparity of Literacy Pattern between Linares E, Bye RA (1987) A study of four medicinal plant Scheduled Castes and Scheduled Tribe Population of Goalpara complexes of Mexico and adjacent United States. Journal of District, Assam. International Journal of Current Research 8: Ethnopharmacology 19:153–83. 30854-30856. Luczaj L, Zovko Končić M, Miličević T, Dolina K, Pandža M (2013) Dogan Y, Nedelcheva A (2015) Wild plants from open markets Wild vegetable mixes sold in the markets of Dalmatia (southern on both sides of the Bulgarian – Turkish border. Indian Journal of Croatia). Journal of Ethnobiology and Ethnomedicine 9: 1-12. Traditional Knowledge 14: 351-358. Martin GJ (1995) Ethnobotany A Methods Manual. vol. 1. Dogan Y, Ugulu I, Durkan N (2013) Wild edible plants sold In the local London: Earthscan Publications. markets of Izmir, Turkey. Pakistan Journal of Botany 45: 177-184. Nicholson MS, Arzeni CB (1993) The market medicinal plants of Giraldo D, Baquero E, Bermúdez A, Oliveira-Miranda MA (2009) Monterrey, Nuevo León México. Economic Botany 47: 184–92. Medicinal plant trade characterization in popular markets of Caracas. Acta Botanica Venezuelica 32: 267–301. Parente CET, da Rosa MMT (2001) Plantas comercializadas como medicinais no Município de Barra do Piraí, RJ. Rodriguésia 52: 47–59. Goyal BR, Agrawal BB, Goyal RK, Mehta AA (2007) Phyto- pharmacology of Moringa oleifera Lam.: An overview. Natural Patiri B, Borah A (2007) Wild Edible Plants of Assam. Guwahati, Product Radiance 6: 347–353. India: Geetakhi Printers and Publishers.

Gupta S, Jain R, Kachhwaha S, Kothari SL (2018) Nutritional and Rabha D (2014) Above ground biomass and carbon stocks of an medicinal applications of Moringa oleifera Lam.—Review of current undisturbed regenerating sal (Shorea robusta Gaertn. F.) forest of status and future possibilities. Journal of Herbal Medicine 11: 1–11. Goalpara district, Assam, Northeast India. International Journal of Environment 3: 147-155. Hanlidou E, Karousou R, Kleftoyanni V, Kokkini S (2004) The herbal market of Thessaloniki (N Greece) and its relation to the Rabha PK (2012) Forest Resource Entrepreneurship: Case study ethnobotanical tradition. . Journal of Ethnopharmacology 91: 281–99. of Goalpara District of Assam. International Journal of Computer Applications in Engineering Sciences, Special Issue on Basic, Hooper D, Field H, Dahlgren BE (1937) Useful Plants and Applied & Social Sciences 2: 361-363. Drugs of Iran and Iraq. Volume 9. Chicago: 7 Field Museum of Natural History. Santos EB, Dantas GS, Santos HB, Diniz MFFM, Sampaio FC (2009) Ethno-botanical studies of medicinal plants for oral Idu McD, Erhabor JO, Efijuemue HM (2010) Documentation on conditions in the municipality of Joao Pessoa, Brazil. Revista medicinal plants sold in markets in Abeokuta, Nigeria. Tropical Brasileira de Farmacognosia 19: 321-324. Journal of Pharmaceutical Research 9: 110–118.

Kasper-Pakosz R, Pietras M, Łuczaj L (2016) Wild and native plants Silalahi M, Walujo EB, Supriatna J, Mangunwardoyo W and mushrooms sold in the open-air markets of southeastern Poland. (2015) The local knowledge of medicinal plants trader and Journal of Ethnobiology and Ethnomedicine 12: 1-17. diversity of medicinal plants in the Kabanjahe traditional market, North Sumatra, Indonesia. Journal of Khanuja SPS (2012) Functional diversity of plant metabolome Ethnopharmacology 175: 432–43. and microbiome in health services to the human life. Proceedings of the National Academy of Sciences, India Section B: Biological Silva MS, Peixoto Al (2009) Abajuru (Chrysobalanus icaco L. Sciences. 82: 291–294. and Eugenia rotundifolia Casar.) commercialized in Rio de Janeiro, Brazil. Revista Brasileira de Farmacognosia. 19: 325-332. Khruomo N, Deb CR (2018) Indigenous wild edible fruits: sustainable resources for food, medicine and income generation – Tibuhwa DD (2018) Edible and Medicinal Mushrooms Sold at a study from Nagaland, India. Journal of Experimental Biology Traditional Markets in Tanzania. Research. Journal of Forestry and Agricultural Sciences 6: 405 – 413. 12: 1-14.

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Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 329 – 334

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

HORMONAL AND IMMUNOLOGICAL CHANGES DURING PREGNANCY PERIOD OF COWS

El-Masry KA1, El-Fouly HA1, Gabr SA*2

1Biological Applications Department, Radioisotope Applications Division, Nuclear Research Center, Atomic Energy Authority, Cairo, Egypt 2Taif University, Khurma University Collage, Kingdom Saudi Arabia

Received – February 05, 2019; Revision – April 04, 2019; Accepted – May 26, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).329.334

KEYWORDS ABSTRACT

Cow The objective of present study was to determine the pattern and impact of pregnancy stage on the concentrations in serum estradiol-17β (E2-17β), testosterone hormones and immunoglobulin G (IgG). Pregnancy For this purpose, blood samples were collected from 16 crossbred, pregnant, lactating cows at the E2-17β intervals of each two weeks. From these collected samples, serum hormone concentration was determined by Radioimmunoassay (125I-RIA) technique and IgG concentration was assayed by using the Testosterone method involves antigen diffusing radially. Results of present study revealed that concentration of E2- 17β is insignificant from 1st to the 4th month of gestation and tended to increase from the 4.5th month to IgG the end of gestation. After parturition E2-17β returned to its normal level on 30th day of postpartum. Further, concentration of testosterone hormone start increasing (29.76 pg/ml) from the 4 th month, after this it gradually increased (P <0.01) with the advances of gestation (185.40 pg/ml) up to the 9 th month, thereafter testosterone level reached to the peak (197.80 pg/ml) at the end of pregnancy as compared to the average during non-pregnancy period. Concentration of IgG remained at basal level from the 1st to 6.5th month of gestation that averaged 23.65 g/l. Thereafter a sharpest increase in IgG level was occurred up to the 8.5th month that followed by a depression in the subsequent period until parturition. It can be concluded that concentrations of E2-17β and testosterone remained at low levels around the 4th – 5th month and progressively increased and showing a sharp rise in the 9.3 month of gestation. Moreover, the levels of IgG insignificantly changed until the 6.5th month of pregnancy and a sharpest rise occurred in the 8.5th month of gestation.

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Hormonal and Immunological Changes during Pregnancy Period of Cows 330

1 Introduction The animals were in moderate milking ability during postpartum period and observed daily for estrus and considered to be in estrus Changes in hormones concentration and functioning during when they stood to be mounted by another cows. Then, jugular pregnancy period of ruminants have been widely studied by blood samples were collected from these non-pregnant cows and various researchers (Al-Anbaky, 2009; Harichadan et al., 2014). were considered as samples for control period. Natural mating Among these, estrogen is essential hormones which help in with pure Brown Swiss bull was carried out for all cows. Records maintain pregnancy and initiation of parturition. Further, of breeding dates were maintained. Blood samples were collected Estradiol-17β stimulates the synthesis of endometrial oxytocin from all mated cows from the 1st days of mating and continued receptors and reduce the rate of LH releases which is must for the throughout pregnancy at the intervals of two weeks. Trans rectal maintain progesterone production during early pregnancy (Peters palpation of the reproductive tract on day 60 of post mating was & Ball, 1995). used as a pregnancy test. The animals were fed on concentrated diets (17% crude protein, 2.5% ether extract and 16.5% crude

The important roles of E2-17β and testosterone hormone during fiber) according to their trimester of pregnancy. The animals had pregnancy period may be attributed to that the biochemical free access to graze green forage throughout pregnancy period. reactions for the formation of these hormones are the same, and Trace elements mixtures (Pfizer Co.) were added daily for all the male sex hormone testosterone can serve as precursor for the animals by 1.5 g/kg dry matter. Drinking water and rice straw synthesis of E2-17β and their pathway involving the conversion of were offered freely ad lib. testosterone to E2-17β is more significant in the ovary (Tietz, The concentrations of E2-17β and testosterone hormones were 1982). Moreover, during pregnancy period, feto-placental tissues determined by radioimmunoassay technique (Elbanna et al., 1988 appears to be the major site of estrogens biosynthesis was able to ) using solid phase coated tubes kits purchased from diagnostic convert testosterone to E2-17β in the basal membrane of theca systems laboratories Inc., Webstir, Texas, USA. Serum IgG cells (Tietz, 1982; Solomon, 1994; Peters & Ball, 1995). Estrogen concentration was measured using kit for IgG of the Binding Sit plays an essential role in the maintenance of pregnancy and Limited Co., Birmingham, UK. initiation of parturition (Peters & Ball, 1995). On the other side, testosterone hormone may modify placental function and had an Statistical analysis of the obtained data were carried out, whereas effect on transport of nutrients to the fetus (Carlsen et al., 2006). the differences between mean values of the items of non- pregnancy period (control period) and each of mean values of Concerning the IgG levels, Janeway et al. (2002) reported that pregnancy period of cows were tested by paired "t" test according IgG is the main immunoglobulin in the blood system accounting to Snedecor & Cochran (1994). for approximately 80% of the total circulating immunoglobulin, whereas its function is to bind pathogenic agents and induce an 3 Results and Discussion immune response. Data presented in Tables 1 and Figure 1 showed that E2-17β According to Harichandan et al. (2014), the relationship between concentrations varied from 32.31 pg/ml (during non-pregnancy testosterone hormone and each of E2-17β and IgG may induce the period) to 37.12pg/ml (in the 4th month of gestation) and the high production of steroid hormones throughout pregnancy difference between these two means was found insignificant. Thereafter, the concentration of E2-17β increased (P<0.01) course, but a wide discrepancy and changes in these items have to 38.67 and 42.64 pg/ml in the 4.5th and 5th month of gestation, been reported by Min et al. (2002), Carlsen et al. (2006) and Al- Anbaky (2009). The present study was therefore undertaken to 300 determine the changes in the general profile of E2-17β, 250 testosterone and IgG concentrations throughout pregnancy as 200 compared with non-pregnancy period of cows. (pg/ml)

150 17β 17β

- 100 2 Materials and Methods 50 0

A total of 16 crossbred (Brown Swiss 50% and Balady 50%) Estradiol pregnant and lactating cows, approximating 4-5 years of age and 0 2 4 6 8 10 weighing 340±20 kg were used for current study and changes in Pregnancy periods (months) E2-17β, testosterone as well as IgG levels during pregnancy Figure 1 Concentration of Estradiol-17β (pg/ml) in non-pregnant and period were studied in detail. during pregnancy periods (months) in Cows

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331 El-Masry et al.

Table 1 Changes in estradiol-17β , testosterone and IgG levels during the pregnancy period of cows

Pregnancy Period Hormonal Concentration (pg/ml)

(Months) Estradiol-17 β Testosterone IgG (g/L) Non-pregnant 32.31±1.68 24.58±1.45 25.41±1.52

1.0 30.18±1.32 22.72±1.12 23.76±1.67 1.5 30.85±0.97 23.41±2.04 25.20±0.97

2.0 34.60±1.02 21.85*±2.33 23.59±1.05

2.5 34.40±1.11 23.31±0.73 25.56±1.36

3.0 28.78±0.89 22.87±0.79 29.01±1.09

3.5 35.71±1.19 23.92±1.03 27.55±1.39

4.0 37.12±1.73 29.76*±1.35 26.10±1.87

4.5 38.69*±1.39 42.25**±1.97 26.28±1.05

5.0 42.67*±2.01 77.32**±2.99 30.43±1.12

5.5 39.34*±2.01 73.82**±3.63 31.53±2.01

6.0 40.95**±3.50 88.8**±4.05 28.01±1.76

6.5 44.25**±2.87 111.5**±5.79 23.65±1.34

7.0 57.9**±2.69 167.73**±6.3 32.61*±2.02

7.5 86.2**±2.57 154.2**±3.26 38.36*±1.39

8.0 58.8**±3.11 162.6**±4.21 43.77**±0.88

8.5 73.2**±3.72 158.9**±3.88 57.79**±1.09

9.0 88.4**±4.18 185.4**±4.95 46.80**±1.13

9.3 248.86**±10.75 197.8**±3.84 34.59*±1.45 *Significant difference at P> 0.05 **Significant difference at P> 0.001

respectively. In the 5.5th month of pregnancy E2-17β pregnant heifers as compared to estrus or other reproductive concentrations increased (P<0.01) progressively with the advance conditions (anestrus or diestrus) and in this situation findings of of gestation and reach maximum (248.86 pg/ml) at the end of present study are in agreement with these earlier reports. pregnancy course. Hirako et al. (2003) reported that during the 1st trimester of Findings of present study are associated with the data of previous gestating cows, E2-17β remained at basal levels until day 80, studies, whereas the concentrations of estrone and E2-17β in followed by an increase in estrone and E217β after day 80 of bovine blood plasma remain low during the early period of gestation. Also, Al-Anbaky (2009) suggested that in pregnant gestation and gradually increased throughout mid, to late gestation cow’s plasma E2 -17β levels varied from 9 pg/ml (in the first third (Hung & Prakash, 1990; Patel, 1999). Similarly, El-Fouly et al. of pregnancy) to 282.60 pg/ml (during the last month of (1998a) showed that E2-17β levels in crossbred cows did not gestation).Similar findings were reported by El-Masry et al. change in the 1st trimester while it significantly increased in the (1997) in a study conducted on Egyptian buffaloes. Several 2nd trimester and increased sharply at the end of the 3rd trimester potential mechanisms affect the level of E2-17β during the of gestation, reached to its peak on day 1 before calving. pregnancy period, whereas the concentration of fetal cortisol from Mekonnin et al. (2017) conducted a study for estradiol level fetal adrenals helps in E2-17β releases from placenta (Gitau et al., detection in pregnant heifers and cows. In both cows and heifers, 2005). Further, the conversion of testosterone hormone into E2- higher estradiol concentrations were recorded during the first 17β held at the granulosa cells by the action of aromatase (Peters trimester of pregnancy as compare to other stages of pregnancy. & Ball, 1995; Hirako et al. 2003). Also, sex of fetus and the levels Similarly, Opara et al. (2006) reported higher level of estradiol in of progesterone hormone might also associate with the releases

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Hormonal and Immunological Changes during Pregnancy Period of Cows 332 and concentration of E2-17β (EL-Fouly et al., 1998 b). The higher 70 levels of E2-17β during third trimester of pregnancy have evoked 60 normal development of mammary alveolar tissues which might be 50 involved in the primary myometrium and triggering the 40 prostaglandin release in farm animals; this would help in further 30 20 myometrium contractions (Peters & Ball, 1995). A combination of 10

all these mechanisms is also conceivable. 0 Immunoglobulin Immunoglobulin (g/L)G Functions of testosterone hormone throughout pregnancy period 0 2 4 6 8 10 have been well reported previously, this hormone induce the Pregnancy periods (months) production of plasma progesterone binding proteins during pregnancy which help in maintaining high progesterone levels Figure 3 Concentration of Immunoglobulin G (g/L) in non-pregnant and during pregnancy periods (months) in cows (Wyk et al., 1994).

Maternal testosterone may also cross the placenta and exert a (1995) and Al-Anbaky (2009). According to these researchers this direct effect on fetal growth and / or energy homeostasis (Carlsen gradually augmentation in the level of testosterone at advance et al., 2006) as well as testosterone with estrogen accelerates stage of gestation may be attributed to the biosynthesis and blood flow to the uterus during parturition (Min et al., 2002). In excretion of testosterone hormone by several sources such as results of present study, testosterone concentration remained at placental tissues, ovary, mammary gland and fetal males. basal levels (23.92 pg/ml) until the 3.5th month of gestation, thereafter testosterone level began to increase (P<0.01) As shown in tables 1 and figure 3, serum IgG levels showed st th progressively with the advance of gestation and a sharpest rise insignificant fluctuation from the 1 month of the 6.5 months recorded 197.80 pg/ml in the 9.3th months of gestation (Tables 1 of gestation. Thereafter, IgG increased sharply in the subsequent th and Figure 2). These results are associated with finding of by months and it reached to the peak in the 8.5 months, followed th th Gaiani et al. (1984) who found that plasma testosterone levels in by the depression in IgG levels in the 9 and 9.3 months of cows ranged between 20 to 50 pg/ml until about 90 days of gestation. General trend of IgG levels throughout pregnancy pregnancy, then it increase progressively to about 220pg/ml period are in agreement with the findings of Carolina et al. around 270 days of gestation. Moreover, Al-Anbaky (2009) (2014) in domestic animals and by Malek et al. (1996) in revealed that testosterone levels in cows were 0.32 ng/ml in the woman; these researchers reported that fetal IgG begins to 3rd month of gestation, increase to 0.541 ng/ml at the end of 8th increase from 13 to 18 weeks of gestation and this improvement month of gestation. Similarly, Emara (2015) reported that non- continuously increases till the end of final trimester and pregnant cows have low level of testosterone (0.08 ng/ml) and typically exceed the maternal IgG concentrations. This increases showed a continuous gradual increase throughout pregnancy in globulin level with the advance of gestation might be due to period till day 3-7 of prepartum (0.28 mg/ml), followed by a drop change in their endocrine profile, since an animal's transition on 30th day of postpartum which averaged 0.097 and 0.06 ng/ml, occurs from non-gravid to gravid state and their transformation respectively. Similar findings were reported by Peters &Ball might have influenced more production of globulin by B- lymphocytes (Harichandan et al., 2014).

250 In addition, high circulating levels of estrogen during pregnancy 200 course (El-Fouly et al., 1998 a; Patel, 1999; Hirako et al., 2003) and glucocorticoids near the onset of parturition can suppress 150 important lymphocyte function (Levkut et al., 2002). Concerning 100 the depression in IgG at postpartum period; Herr et al. (2011) th 50 revealed that plasma IgG level recovered by the 4 week of postpartum and the extent of IgG reduction seemed to be 0 Testosterone (pg/ml) Testosterone depended on the initial IgG concentration. Such reduction in 0 2 4 6 8 10 immunoglobulin concentration at the terminal stage of pregnancy pregnancy periods (months ) in cows might have occurred because the transition of globulin

Figure 2 Concentration of Testosterone (pg/ml) in non-pregnant and fraction from the blood to the mammary gland and tubular genital during pregnancy periods (months) in cows. tract (Liberg,1977; Harichandan et al., 2014).

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From the results of present study it can be concluded that Harichandan PP, Mohanty DN, Das S, Patra BK (2014)Hormonal interaction and changes in the levels of E2-17 β and testosterone and immunological studies in different trimester of pregnancy in hormones were augmented and coincided with the advance of cows. Indian Journal of Animal Reproduction 35: 42-44. gestation for sustenance of pregnancy, showing higher concentrations during the late months of pregnancy compared to Herr M, Bosdedt H, Failing K (2011) Ig G and Ig M levels in non -pregnancy ones, and the levels of IgG insignificantly changed dairy cows during the periparturient period. Theriogenology 15: until the 6.5th months of pregnancy and a sharpest rise occurred in 377-385. the 8.5th months of gestation. Hirako M, Takahashi T, Takahashi H, Patel O, Domeki I (2003) Conflict of Interest Changes in plasma estrogen concentrations during the first trimester of gestation in dairy cows: comparison with the origin of The authors declare that there is no conflict of interest in this manuscript. embryos and featal number. Japan Agricultural Research Quarterly 37: 195-200. References Hung NN, Prakash BS (1990) Influence of gestation on blood Al- Anbaky KIH (2009) A study of serum steroid hormones plasma concentration of oestrone and oestrone sulphate in karan concentration of pregnant cows. Iraqi Journal of Veterinary swiss cows and Murrah buffuloes. Brazilian Veterinary Journal Medicine 33: 180 – 182. 146: 449-456.

Carolina L, Rodrigues MN, Favaron PO, Miglino MA (2014) Janeway CA, Travers P, Walport M, Shlomchik M (2002) Immunoglobulin transport during gestation in domestic animals and Immunobiologia: O sistemaimunenasaude e nadoenca. Artmed, humans: A review. Open Journal of Animal Sciences 4:323-336. Porto Alegre.

Carlsen SM, Jacobsen G, Romundstad P (2006) Maternal Levkut M, Pistl J, Revajova V, Choma J, Levkutova M, David testosterone levels during pregnancy are associated with offspring V(2002) Comparison of immune parameters in cows with normal size at birth. European Journal of Endocrinology.155:365-370. and prolonged involution time of uterus. Veterinarni Medicina - UZPI (Czech Republic) 47 : 277-282. Elbanna IM, El-Asrag HA, Gamal MH (1988) An improved method for estradiol-17β radioimmunoassay. Isotope and Liberg P (1977) Agarose gel electrophoretic fractionation of Radiation Researches 20: 141-144. serum proteins in adult cattle. A study of clinically healthy cows. Acta Veterinaria Scandinavica 18:40-53. El-Fouly HA, El- Masry KA, Gamal MH (1998a) Maternal body weight during trimester of pregnancy as an indicator of the Malek A, Sager R, Kuhn P, Nicolaides KH, Schneider H (1996) development of male and female fetuses and their relationship Evolution of Maternofetal transport of immunoglobulins during with some steroid hormones and blood biochemical levels in human pregnancy. American Journal of Reproductive Baladi cows. Zagazig Veterinary Journal 26: 56-68. immunology, 36:248-255.

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Hormonal and Immunological Changes during Pregnancy Period of Cows 334

Patel OV (1999) Plasma oestrone and oestradiol concentrations The Iowa state University press, Ames, USA. throughout gestation in cattle: relationship to stage of gestation and fetal number. Research in Veterinary Science 66:129-133. Solomon S (1994) The primate placenta as an endocrine organ: steroids. In: Knobil E, Neill JD (Eds.), The physiology of Peters AR, Ball PJH (1995) parturition and lactation. In: Reproduction reproduction, Raven Press, New York, USA, Pp. 863-873. in Cattle, 2ndedn, Blackwell Science Ltd, UK, Pp. 127-144. Tietz NW (1982) Fundamental of clinical chemistry. In: Norbert Gaiani R, Chiesa F, Mattioli M, Nannetti G, Galeati G (1984) WT (Ed.), Sounders Company, Philadelphia. Androstenedione and testosterone concentrations in plasma and milk of cow throughout pregnancy. Journal of Reproduction and Wyk VN., Van Ande RJ, Lauw AI (1994) Concentrations of Fertility 70: 55-59. plasma total and unbound progesterone and testosterone during pregnancy in Cape Porcupines. Comparative Biochemistry and Snedecor GW, Cochran WG (1994) Statistical Methods. 8th Ed., Physiology 108A:265-271.

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Journal of Experimental Biology and Agricultural Sciences, June - 2019; Volume – 7(3) page 335 – 342

Journal of Experimental Biology and Agricultural Sciences

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ISSN No. 2320 – 8694

In vitro CYTOTOXIC EFFECTS OF HYBRID GRAPHENE OXIDE NANOCOMPOSITES (hGONCs) ON CRYOPRESERVED CAPRINE WHARTON’S JELLY DERIVED MESENCHYMAL STEM CELLS (WJ-MSCs)

Sandeep A. Dhenge1*, Nitin E. Gade2, Omprakash Mishra2, Mangesh M. Vaidya1

1Department of Veterinary Physiology, College of Veterinary & Animal Sciences (MAFSU, Nagpur), Udgir, Dist. Latur (M.S.) India-413 517 2Department of Veterinary Physiology& Biochemistry, College of Veterinary Science & Animal Husbandry (CGKV, Durg), Anjora, Durg (C.G.) India-491 001

Received – February 11, 2019; Revision – April 03, 2019; Accepted – May 16, 2019 Available Online – June 10, 2019

DOI: http://dx.doi.org/10.18006/2019.7(3).335.342

KEYWORDS ABSTRACT

Nanotechnology In present study, caprine Wharton’s jelly derived mesenchymal stem cells (WJ-MSCs) were isolated and characterized by osteogenic and adipogenic differentiation. Total 12 hybrid graphene oxide Graphene nanocomposites (hGO NCs) such as N2 doped GO-HA NCs, P doped GO-HA NCs, S doped GO-HA

Cryopreservation NCs, N2 doped GO-SiO2 NCs, P doped GO-SiO2 NCs, S doped GO-SiO2 NCs, N2 doped GO-TiO2 NCs, P doped GO-TiO2 NCs, S doped GO-TiO2 NCs, N2 doped GO-Au NCs, P doped GO-Au NCs and S Caprine doped GO-Au NCs were incorporated at doses 100, 50, 25 , 10 and 0 µg/ml in vitrification solution. Caprine WJ-MSCs were cryopreserved by using open pulled straw (OPS) vitrification method and Stem cells analysed hGO NCs effect on their post thaw viability and culture characteristics. Caprine WJ-MSCs were exhibited normal fibroblastoid morphology and differentiated in to osteogenic and adipogenic lineages. The significant (P<0.01) highest and lowest caprine WJ-MSCs post thaw viability

(cumulatively) was observed in P-GO-TiO2 NC and P-GO-HA NC groups, respectively along with all doses (cumulatively) significantly (P<0.01) decreased post thaw cell viability as compared with control. All hGO NCs were significantly (P<0.01) decreased caprine WJ-MSCs post thaw viability at doses 100 and 50 µg/ml as compared to 25, 10 and 0 µg/ml doses. Post thaw caprine WJ-MSCs were grew in normal pattern with similar fibroblast like morphology on days 14. In conclusion, all hGO NCs at doses

50 and 100 µl/ml are cytotoxic and P-GO-TiO2 NC is least decrease caprine WJ-MSCs post thaw viability.

* Corresponding author All the articles published by Journal of Experimental Biology and Agricultural Sciences are licensed under a E-mail: [email protected] (Sandeep A. Dhenge) Creative Commons Attribution-NonCommercial 4.0

International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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In Vitro Cytotoxic Effects Of Hybrid Graphene Oxide Nanocomposites (Hgoncs) On Cryopreserved Caprine Wharton’s Jelly Derived Mesenchymal Stem Cells (Wj-Mscs) 336

1 Introduction GO hybridization or doping with organic or inorganic

nanomaterials or nanoparticles exploit their unique properties Nanotechnology is the study of materials and structures which are (Wang et al., 2012) and biocompatibility (Nishida et al., 2014) in manipulate at nanoscale level and usually, a deliberately stem cells. In vitro maintenance of stem cells is difficult task as it engineered material with at least one dimension 1-100 nm is changes in its genotype and phenotype, lead to senescence and called as nanomaterial (Hornyak et al., 2008). It is applied in transformation as well as contaminated in incubator. Hence, stem different fields such as chemistry, energy, textile, cosmetics, space cells cryopreservation technique can provide potential and regular research, information technology, biomedicine and agricultural. stem cells source, save time and also culture materials. Stem cells Recently various types of nanomaterials are widely used in were cryopreserved using vitrification by opened pulled straw biomedicine for drug discovery (Estelrich et al., 2015) and (OPS) method (Bahadori et al., 2009), but post thaw survival rate delivery (Ambwani et al., 2018), bio-imaging (Lobatto et al., is so variable, however, nanoparticles incorporation in 2012), tissue engineering and regenerative medicine (Jayakumar cryoprotectant solution might improve efficiency of vitrification et al., 2011), proteomics (Patil et al., 2015), genomics (Basu et al., solution by changing its chemical properties (Li et al., 2016; Xu et 2013), in vitro disease diagnosis (Mamaeva et al., 2018) along al., 2016). However, best of our knowledge, invitro efficacy of with treatment (Kamaly et al., 2016) as well as in stem cell hybrid graphene oxide (hGO) as GO doped with nitrogen (N2), research (Deb et al., 2012). In stem cell research, nanotechnology phosphorus (P) and sulphur (S) and their nanocomposites with is applied in stem cell culture (Goto et al., 2007), expansion, hydroxyapatite (HA), Silica (Si), Titanium oxide (TiO2) and gold differentiation and transplantation (Guo et al., 2017), nano (Au) were not studied instem cells. hGO NCs may be useful in delivery of DNA, RNAi and proteins in stem cells differentiation stem cell research, regenerative medicine and also stem cells post (Park et al., 2011) as well as in vivo delivery (Nguyen, 2013) and thaw survivability rate can be increased by their incorporation in imaging of stem cells (Michalet et al., 2005). Stem cells are vitrification solution. Hence, in vitro efficacy of hGONCs in specialized progenitor cells residing in nearly all organ tissue and animal stem cell model is an important and therefore, present have enormous potency to self-renew and repairs damage cells study was carried with the objective to study comparative in vitro and differentiate in vitro into their lineage cells. Mesenchymal effect of hGO NC soncaprine WJ-MSCs by studying post thaw stem cells (MSCs) are adult stem cells and can be isolated from cell viability. bone marrow and fetal adnexa tissue and differentiate into osteocytes, adipocytes and chondrocytes (Pittenger et al., 1999), 2 Materials and Methods pancreatic cells (Saman et al., 2014), hepatocytes (Borhani et al., Present study was conducted in Department of Veterinary 2015) and were used to treat numerous regenerative diseases Physiology and Biochemistry, College of Veterinary Science and (Sangeetha et al., 2017). Fetal adnexa derived MSCs can be Animal Husbandry, Anjora, Durg (C.G.)-491 001 India and study isolated easily and Umbilical cord Wharton’s jelly derived MSCs protocol approved by Institution Animal Ethical Committee (No. (WJ-MSCs) expand rapidly and differentiate into osteocytes, VPB/PhD-1/2017). adipocytes and chondrocytes (Somal et al., 2016; Somal et al., 2017). Carbon-based nanomaterials (CBNs) are considered as 2.1 Isolation of caprine WJ-MSCs excellent nanomaterials in tissue engineering (Oprych et al., 2016) and other biological applications (Yang et al., 2007) due to their Gravid caprine uteri (45-60 days) were collected from nearby relative biocompatibility. Graphene (GN) is an atom thick pure slaughter house and uteri were transported within 1 hr to carbon monolayer arranged in two-dimensional honeycomb Department of Veterinary Physiology and Biochemistry, College of structure (Allen et al., 2010) and its derivatives as graphene oxide Veterinary Science and Animal Husbandry, Anjora, Durg, (GO) and reduced graphene oxide (rGO) were applied in Chhattisgarh, India. Uteri were washed thoroughly by sterile saline biotechnology (Kim et al., 2011). However, dose and exposure and aseptically 2-3 cm long umbilical cord was cut and proceeded to time depended cytotoxicity of hybrid graphene nanomaterials i.e. isolate WJ-MSCs (Babaei et al., 2008). Caprine WJ-MSCs were graphene-based nanomaterials (GBNs) was reported in A549 lung cultured in Dulbecco’s modified eagles’ medium (DMEM) fortified epithelial cells (Wadhwa et al., 2011), 264.7 RAW macrophage with 15% Fetal Bovine Serum (FBS) and kept in CO2 incubator at 0 (Szczypta et al., 2012; Figarol et al., 2015) and caprine WJ-MSCs 37 C with 5% CO2. Cells were observed periodically under inverted (Dar et al., 2015; Gade et al., 2015). In spite this, certain GBNs microscope and culture media was changed every after 4th days and were showed biocompatibility in human osteoblast (Baradaran et cells were passaged on day 14 and third passaged caprine WJ-MSCs al., 2014), mouse osteoblastic MC3T3-E1 cells (Nishida et al., were characterized with Alzarin red and Oil red O staining 2014), caprine BM-MSCs (Elkhenany et al., 2015) and murine (Baghaban et al., 2008) to test their osteogenic and adipogenic MSCs (Kim et al., 2018) in dose dependent manner. In addition, differentiation potential, respectively.

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337 Dhenge et al.

2.2 Hybrid graphene oxide nanocomposites (hGO NCs) The third passage caprine WJ-MSCs were induced with respective differentiation media for 21 days to differentiate into Total 12 hGO NCs such as N2 doped GO-HA NCs, P doped GO- osteocytes and adipocytes and stained positively with Alzarine red HA NCs, S doped GO-HA NCs, N2 doped GO-SiO2NCs, P doped (figure 2A) and Oil red O staining(figure 2B), respectively. GO-SiO2NCs, S doped GO-SiO2NCs, N2 doped GO-TiO2NCs, P doped GO-TiO2NCs, S doped GO-TiO2NCs, N2 doped GO-Au NCs, P doped GO-Au NCs and S doped GO-Au NCs were generously supplied by Department of Physics, BHU, Banaras, Uttar Pradesh (India). hGO NCs were sterilized and total 5 treatment groups with different doses (100 µg/ml, 50 µg/ml, 25 µg/ml, 10 µg/ml and 0 µg/ml) were constituted to study effect of hGO NCs on caprine WJ-MSCs post thaw viability.

2.3 Cryopreservation of caprine WJ-MSCs

Third passaged caprine WJ-MCSs were cryopreserved with hGO NCs Figure 2 Characterization of WJ-MSCs [A.] Alzarin red staining by using open pulled straw (OPS) vitrification method (Vajta et al., and [B.] Oil red O staining 1997; Vajta et al., 1998) with some modifications. Cells were harvested and cryopreserved by using equilibration [10 % Ethylene Post thaw viability of caprine WJ-MSCs with hGO NCs in Glycol (EG) + 10 % Dimethyl Sulphoxide (DMSO)] and vitrification vitrification solution was analyzed after 7 days of cryopresrvation. (20 % EG + 20 % DMSO + 0.5 M / L sucrose) solutions in DMEM The cumulative significant (P<0.01) highest and lowest caprine supplied with 20% FBS. hGO NCs were added in vitrification WJ-MSCs post thaw cell viability was observed in P-GO-TiO2 solution as per designed doses to study cytotoxicity. Caprine WJ- NC and P-GO-HA NC added groups, respectively among all hGO MSCs transferred in media and suspended in equilibration solution for NCs (Table 1). In addition, caprine WJ-MSCs post thaw viability 5 minute and then vitrification solution mixed for 30 - 40 second. significantly (P<0.01) decreased in all doses of hGO NCs Cells droplets were loaded in sterilized French pulled straws by (cumulatively) as compared with control however, 100 and 50 capillary effect and submerged immediately into liquid nitrogen and µg/ml doses significantly (P<0.01) decreased post thaw cell stored for 7 days. Cells were thawed and suspended in DMEM with viability as compared with 25and 10 µg/ml (Table 2). 15% FBS and cell viability was assessed (Bregoli et al., 2009).

2.4 Statistical analysis Table 1 Cumulative effect of each hGO NCs on caprine WJ- MSCs post thaw viability

The data recorded in present study was analyzed with Full Factorial Sr. No. hGO NCs Post thaw viability % design and One way - ANOVA (IBM SPSS Statistics 25 software) 1. N -GO-HA NC 50.40±1.74abc and values are expressed as mean ± standard error (S. E.) and P<0.01 2 and P < 0.05 are considered to be statistically significant. 2. P-GO-HA NC 45.93±2.29a abc 3 Results 3. S-GO-HA NC 49.93±2.40 abc 4. N2-GO-SiO2 NC 50.53±1.63 Caprine WJ-MSCs are plastic adherent and exhibited bc heterogenous morphology however, mostly fibroblastoid like cells 5. P-GO-SiO2 NC 51.33±1.69 abc were observed and reached confluent stage on day 14 (Figure 1). 6. S-GO-SiO2 NC 49.53±1.90

ab 7. N2-GO-TiO2 NC 46.60±1.78

c 8. P-GO-TiO2 NC 51.53±2.02

abc 9. S-GO-TiO2 NC 46.93±2.05

ab 10. N2-GO-Au NC 46.53±2.47

11. P-GO-Au NC 46.60±2.50ab

12. S-GO-Au NC 47.87±2.59abc

Number of replicate =15; Mean values bearing superscript in Figure 1 Caprine WJ-MSCs in confluent stage on day 14 column differed significantly from each other (P<0.01)

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In Vitro Cytotoxic Effects Of Hybrid Graphene Oxide Nanocomposites (Hgoncs) On Cryopreserved Caprine Wharton’s Jelly Derived Mesenchymal Stem Cells (Wj-Mscs) 338

Table 2 Cumulative effect of each dose of hGO NCs on caprine WJ- However, each hGO NC significantly (P<0.01) decreased

MSCs post thaw viability caprine WJ-MSCs post thaw viability in doses 100 and 50 Sr. No. Dose (µg/ml) Post thaw viability % µg/ml as compared to 25, 10 and 0 µg/ml doses which non- significantly differed (Table 3). Here, it showed that, each 1. 100 35.42±0.71a hGO NC in doses 25 and 10 µg/ml vitrification solution not b 2. 50 42.36±0.81 caused any significant cytotoxic effect on caprine WJ-MSCs

c post thaw viability. 3. 25 53.94±0.61

4. 10 53.69±0.53c Post thaw caprine WJ-MSCs were grew in colonies and observed elongated morphology typical as fibroblast cells and only minute 5. 0 57.81±0.46d cells were floated in media and detached from surfaces and

Number of replicate =36; Mean values bearing superscript in column observed confluent monolayer on days 14th (Figure 3). differed significantly from each other (P<0.01) 4 Discussion

The aim of the present experiment was to study comparative effect of hGO NCs on caprine WJ-MSCs post thaw viability. Caprine WJ-MSCs were successfully isolated and cultured till third passage and phenotypically characterized by Alzarin red and Oil red O staining and confirmed their osteocytes and adipocytes differentiation capability, respectively (Figure 2). Caprine WJ- MSCs are fibroblast like cells which differentiated into osteocytes, adipocytes and chondrocytes (Somal et al., 2016) and cryopreservation did not alter their stemness but, post thaw cell

Figure 3 Caprine WJ-MSCs post thaw morphology on day [A.] viability was reduced (< 60 %) as like present study in control 12th and [B.] 14th

Table 3 Effect of hGO NCs on caprine WJ-MSCs post thaw viability (n=3, P<0.01)

Post thaw cell viability % at different doses (μg / ml) Sr. No. hGO NCs 100 50 25 10 0

a b bc bc c 1. N2-GO-HA NC 37.67±2.40 47.67±2.03 54.33±3.71 54.67±1.45 57.67±2.03

2. P-GO-HA NC 32.33±2.96a 38.00±1.53a 51.33±3.38b 50.67±3.38b 57.33±2.73b

3. S-GO-HA NC 34.00±2.08a 42.67±2.03b 57.00±1.15c 55.33±1.76c 60.67±1.45c

a ab bc bc c 4. N2-GO-SiO2 NC 39.67±4.63 47.67±2.96 52.00±3.79 54.33±1.86 59.00±1.73

a b c c c 5. P-GO-SiO2 NC 40.00±1.53 46.33±0.88 55.67±2.33 56.33±1.45 58.33±1.76

a b c bc c 6. S-GO-SiO2 NC 36.00±2.89 45.67±3.76 55.67±2.03 54.00±2.65 56.33±1.45

a a c c c 7. N2-GO-TiO2 NC 36.00±1.73 40.67±1.76 50.67±2.60 50.33±2.67 55.33±2.03

a b c c c 8. P-GO-TiO2 NC 35.67±2.03 48.00±2.65 56.33±2.40 58.67±2.03 59.00±2.31

a a b b b 9. S-GO-TiO2 NC 35.00±3.79 39.00±3.46 50.67±1.45 52.00±2.08 58.00±2.65

a a b b b 10. N2-GO-Au NC 33.33±2.60 38.33±1.86 54.33±2.73 49.00±5.03 57.67±2.96

11. P-GO-Au NC 30.00±3.06a 39.00±4.04b 55.67±1.45c 52.67±0.88c 55.67±1.45c

12. S-GO-Au NC 35.33±3.84a 35.33±2.33a 53.67±2.03b 56.33±0.88b 58.67±1.20b

Mean values bearing superscript in row differed significantly from each other.

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339 Dhenge et al. group (Somal et al., 2017). However, hWJ-MSCS post thaw addition, in support to present study, similarly GO and GO-SiO2 viability not significantly differed and observed 95±2.3% survival nanoparticles significantly increased human neuronal stem cells rate and fibroblast like morphology (Massood et al., 2013) which (hNSCs) viability with highest expression of neuronal (TuJ1, is also justified in present study that, caprine WJ-MSCs post thaw MAP2 and Synapsin) and axonal (GAP43) markers (Solanki et morphology did not change and cells typically showed their al., 2013). Similarly, GO filler promotes proliferation in L929 and fibroblast like morphology. In addition, post thaw rat BM-MSCs MG63 cell lines but concentration of GO coatings determined in were formed colonies and showed fibroblastic or spindle shaped vitro biocompatibility of Ti and HA nano sheets (Li et al., 2014). morphology and post thaw cell viability for vitrification method However, core shell fluorescently labelled 15, 60 and 200 nm size and OPS vitrification method observed as 81.33±6.93% and 80.83 SiO2 nanoparticles exhibited biocompatibility at doses 10, 20, 30, ±6.4 %, respectively (Bahadori et al., 2009). GBNs such as 40 and 50 μg/ml in THP-1 derived macrophages, A549 epithelial graphene quantum dots (Dar et al., 2015), GO-Fe2O3 NCs (Gade cells, HaCaT keratinocytes and NRK-52E kidney cells (Hsiao et et al., 2015) and N2-GO-HA NCs (Dhenge et al., 2018) were al., 2014). In addition, ~50 nm, ~200 nm and ~400 nm size core- exhibited dose (10, 25, 50 and 100 µg/ml) dependent cytotoxicity shell fluorescent SiO2 nanoparticlesdid not decreased human in caprine WJ-MSCs. However, nanoparticles incorporation in MSCs viability at concentration 10, 50, 100 and 250 μg/ml (Yang cryoprotectant solutions can improve potency of vitrification et al., 2017). Also, TiO2, SiO2 and multi-walled carbon nanotubes solution (Li et al., 2016) by changing its chemical properties (Xu (MWCNTs) were exhibited cytotoxicity in 3T3 fibroblast, RAW et al., 2016) and this is justified in present study, as all hGO NCs 264.7 macrophage and human bronchiolar epithelial cells (hT) in in doses 10 and 25 µl/ml did not significantly (P<0.01) reduced 100 and 1000 μg/ml and SiO2 exhibited highest cytotoxicity in caprine WJ-MSCs post thaw viability as compared to control RAW cells (Sohaebuddin et al., 2010). These findings are not in

(Table 3). But, each hGO NCs in 50 and 100 µl/ml doses were agreement with the results of present study where N2, P and S significantly (P<0.01) reduced caprine WJ-MSCs post thaw doped hybrid GOs with SiO2 and TiO2 nanoparticles significantly viability as compared to 0, 10 and 25 µl/ml doses. Hence, in (P<0.01) decreased caprine WJ-MSCs post thaw viability in 50 present study it was confirmed that, each hGO NC in doses 10 and and 100 μg/ml doses (Table 3) and this difference in cell viability

25 µl/ml in vitrification solution did not cause any cytotoxicity in might be due to difference in vitro cell models. However, SiO2 caprine WJ-MSCs during cryopreservation. Here, GO in each coated HA triphasic (Ca-66.36%, P-25.33% and Si-8.29%) bio- hGO NC reduced its cytotoxicity slightly which is in agreement composite (HA-SiO2) scaffolds (0.3x0.2x0.1 cm dimension) were with earlier findings as significantly (P<0.01) increased human showed biocompatibility in rabbit BM-MSCs (Ravindran et osteoblast viability and alkaline phosphatase activity (ALP) that al.,2016) which is as supportive to present study findings in doses cultured on HA-GN NCs powder deposited on Ti substrates (Liu 10 and 25 μg/ml. et al., 2014). But, high GO concentration (0.5 wt. %) in film suppressed cell spreading and significantly inhibited both Conclusion proliferation and ALP activity of MC3T3-E1 cells (Nishida et al., The present study concluded as, caprine WJ-MSCs are fibroblast 2014). However, caprine BM-MSCs cultured on GO coated tissue like cells and differentiate into osteocytes and adipocytes. Post culture plates exhibited fibroblast like typical MSCs morphology, thaw caprine WJ-MSCs viability is decrease by hGO NCs at grew in clusters (200-250), >95 % viability with 24 hrs population higher doses (100 and 50 µl/ml) and amongst all hGO NCs, P- doubling time and observed similar cellular proliferation in plane GO-TiO NC is least cytotoxic and it can be use at lower doses as well as GO coated tissue culture plates (Elkhenany et al., 2015) 2 (25 and 10 µl/ml)in stem cell research. which might be justified in present study as GO absolutely enhanced biocompatibility of each hGO NC in 10 and 25 µg/ml Acknowledgement doses by showing similar caprine WJ-MSCs post thaw viability (Table 3). However, cumulatively, significant (P<0.01) lowest and All the authors are thankful to The Dean, College of Veterinary highest caprine WJ-MSCs post thaw viability was observed in P- Science & A.H., Anjora, Dist. Durg for providing fund and

GO-HA NC and P-GO-TiO2 NC incorporated groups, respectively laboratory facilities to conduct present study. and this might be due to HA (Baradaran et al.,2014) and TiO2 nanoparticles (Salla et al.,2013) which significantly decreased and Conflicts of interest increased human osteoblast cell viability and MSCs proliferation, None declared. respectively than control. Hybridization of GN and GO with bio- mineral like HA increased mouse osteoblast (MC3T3-E1) cell References viability (Kim et al., 2011) and also Au-GO entered in cytoplasm and increased enhanced Raman signals in HeLa cells with Allen MJ, Tung VC, Kaner RB (2010) Honeycomb carbon: A increased survivability and proliferation (Liu et al., 2012). In review of graphene. Chemical Reviews110:132-145.

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